CYP1B1 nucleic acids and methods of use

ABSTRACT

The present invention provides nucleic acids containing transcriptional units that encode CYP1B1 polypeptides or portions thereof, wherein the transcriptional units lack sequences found in the untranslated region (UTR) of naturally occurring forms of the CYP1B1 transcript. The nucleic acids of the invention lack translational repressor elements and thus provide for a system of enhanced translation of the CYP1B1 polypeptide or portions thereof. Also disclosed are methods of administering nucleic acids to a mammal and use in the treatment of proliferative disorders or cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/244,501, filed Oct. 31, 2000, U.S. ProvisionalApplication No. 60/261,719, filed Jan. 12, 2001, and U.S. ProvisionalApplication No. 60/298,428, filed Jun. 15, 2001. These applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The invention relates to CYP1B1 nucleic acids and methods of useto induce an immune response.

BACKGROUND OF THE INVENTION

[0003] Cytochrome P450 constitutes a large gene family of enzymes thatparticipate in the oxidative activation and/or deactivation of a widerange of xenobiotics, including many potential carcinogens and severalanticancer drugs (Guengerich and Shimada (1991) Chem. Res. Toxicol.4:931; Gonzalez and Gelboin (1994) Drug Metab. Rev. 26:165; Kivisto etal. (1995) Br. J. Clin. Pharmacol. 40:523).

[0004] The human CYP1 gene family, one of the major P450 families,consists of three individual forms classified into two sub-families.CYP1B1, a member of one sub-family, is 543 amino acids in length. It isstructurally distinct from the two members of the CYP1A2 subfamily (Tanget al., J. Biol. Chem. (1996) 271:28324).

[0005] Studies of various types of cancer, including breast cancer,esophageal cancer and soft tissue sarcomas, have shown that there may betumor-specific expression of a CYP1B1 form of P450 (see Murray et al.(1991) Br. J. Cancer 63:1021; Murray et al. (1993) J. Pathol. 171:49;Murray et al. (1994) Gut 35:599). Immunohistochemistry studies of CYP1B1show a strong immunoreactivity for several different types of tumors(bladder, breast, colon, kidney, lung, esophagus, ovary, skin, stomach,uterus, bone and connective tissue, lymph node, brain, and testis) (seeWO 97/12246, herein incorporated by reference).

SUMMARY OF THE INVENTION

[0006] The invention is based on the discovery that nucleic acids can beconstructed that contain transcriptional units that encode CYP1B1polypeptides or portions thereof, wherein the transcriptional units lacktranslational repressor elements. These elements may be located in theuntranslated region (UTR) of naturally occurring forms of the CYP1B1transcript, or in the coding sequence, or both. The nucleic acids of theinvention lack some or all of the CYP1B1 endogenous translationalrepressor elements and thus provide for a system of enhanced translationof the CYP1B1 polypeptide or portions thereof. The nucleic acids of theinvention can also contain mutations, deletions, insertions, orrearrangements that promote the immunogenicity of the encoded protein.The polypeptides encoded by the nucleic acids described herein areuseful for stimulating an immune response in a mammal.

[0007] In one aspect, the invention features a nucleic acid including atranscriptional unit that contains a coding sequence that encodes apolypeptide containing CYP1B1 or a portion thereof that contains apeptide that binds to an MHC class I or class II molecule or is a B cellepitope. The transcriptional unit does not contain a translationalrepressor element operably linked to the coding sequence.

[0008] As used herein, a “transcriptional unit” refers to a nucleic acidcontaining a translation start signal, followed by an open reading frameoptionally including an intron and appropriate splice donor and acceptorsites, followed by a termination codon, wherein the nucleic acid iseither (1) an RNA or (2) a sequence of nucleotides that is transcribedinto an RNA. A “translation start signal” refers to an initiation codonin the context of a Kozak consensus sequence. A “translational repressorelement” refers to a nucleotide sequence located in the untranslatedregion of a transcript that, when present, decreases the level oftranslation of a polypeptide encoded by the transcript by at least 25%relative to the transcript lacking the nucleotide sequence. Atranslational repressor element can cause a decreased level oftranslation by, for example, preventing ribosome binding to a transcriptor decreasing the half life of a transcript.

[0009] A polypeptide encoded by a nucleic acid described herein containsa segment of CYP1B1 that is at least eight amino acids in length. In oneexample, the polypeptide contains the sequence FLDPRPLTV (SEQ ID NO:22).In another example, the polypeptide contains the sequence of any of SEQID Nos:31-39. In another example, the polypeptide is less than 400, 300,200 or 100 amino acids in length.

[0010] As used herein, a “segment” is an amino acid sequence which (a)corresponds to the sequence of a portion (i.e., fragment less than all)of a CYP1B1 protein and (b) contains one or more epitopes. For clarity,the term “segment” is used herein to denote a part of a polypeptideencoded by a nucleic acid of the invention, while the term “portion” isused to denote the corresponding part of the naturally occurringprotein. By “epitope” is meant a peptide which binds to the bindinggroove of an MHC class I or class II molecule or to the antigen-bindingregion of an antibody. A methionine codon can be included at the 5′ endof this or any other coding sequence of the invention, to facilitatetranslation. In addition, a polypeptide encoded by a nucleic aciddescribed herein can encode a targeting signal, as described in moredetail below.

[0011] A transcriptional unit described herein can contain an RNAstabilization sequence. “RNA stabilization sequence” refers to anucleotide sequence located in the untranslated region (UTR), 5′ or 3′UTR or both, of a transcript that, when present, increases the half lifeof the transcript relative to a transcript lacking the nucleotidesequence.

[0012] A nucleic acid described herein can contain an inducible promotersequence operably linked to the transcriptional unit. “Induciblepromoter sequence” refers to a sequence of nucleotides, wherein thebinding of an agent, e.g., a metal or some other non-proteinaceouscompound, to the sequence of nucleotides results in enhancedtranscription of the transcriptional unit to which the sequence ofnucleotides is operably linked. An example of an inducible promotersequence is the metallothionine promoter.

[0013] A polypeptide encoded by a nucleic acid of the invention mayoptionally include a targeting signal. A targeting signal is a peptidewhich directs intracellular transport or secretion of a peptide to whichit is attached. The targeting signal can be at the amino terminus, e.g.,a signal sequence, or carboxy terminus, or within the hybridpolypeptide, so long as it functions in that site.

[0014] The targeting signal can be, for example, a signal sequence. Anysignal sequence that directs the encoded protein to the endoplasmicreticulum and/or causes secretion of the encoded protein to which it isattached is suitable. A preferred targeting signal is the signal peptideof HLA-DRα: MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:27). Another signalsequence that can be linked to a polypeptide described herein has thefollowing sequence: MAISGVPVLGFFIIAMLMSAQESWAPRAT (SEQ ID NO:40).Another signal sequence that can be linked to a polypeptide describedherein is the E1A signal sequence.

[0015] The targeting signal may optionally be modified to introduce anamino acid substitution at the junction(s) between the targeting signaland the adjacent segment(s) to promote cleavage of the targetingsequence from the epitopes by, e.g., a signal peptidase.

[0016] In another aspect, the invention features a nucleic acidincluding a transcriptional unit containing a coding sequence thatencodes a polypeptide containing CYP1B1 or a portion thereof thatcontains a peptide that binds to an MHC class I or class II molecule,wherein the transcriptional unit does not contain 150 consecutivenucleotides of SEQ ID NO:18 or SEQ ID NO:19. Preferably the CYP1B1 orportion thereof corresponds to the sequence of a naturally occurringCYP1B1 polypeptide of a mammal, e.g., a human.

[0017] In one example, a transcriptional unit does not contain at leastone of SEQ ID NOs:3-9 or 15-17. In another example, the transcriptionalunit does not contain 50, 25, or 10 consecutive nucleotides of SEQ IDNO:18 or SEQ ID NO:19. In another example, the transcriptional unit doesnot contain any of SEQ ID NOs:3-9 or 15-17.

[0018] A transcriptional unit can contain a translational regulatorysequence operably linked to the coding sequence. “Translationalregulatory sequence” refers to a sequence of nucleotides, wherein thebinding of an agent to the sequence of nucleotides results in enhancedtranslation of a polypeptide encoded by the coding sequence to which thesequence is operably linked. In one example, the translationalregulatory sequence is an iron responsive sequence.

[0019] In another aspect, the invention features a nucleic acid thatcontains a transcriptional unit that encodes a hybrid polypeptidecontaining a first and a second segment of CYP1B1. The first and secondsegments are either contiguous or separated by a spacer amino acid orspacer peptide. The first and second segments are each at least eightamino acids in length and are non-contiguous portions of CYP1B1.

[0020] By “spacer amino acid” is meant a single residue inserted betweentwo neighboring segments (“A” and “B”, in that order) in a polypeptideof the invention, where the residue is different from the amino acidwhich flanks the carboxy terminus of A and also is different from theamino acid which flanks the amino terminus of B in the full lengthCYP1B1 protein. Thus, the spacer amino acid forms a point ofdiscontinuity from the CYP1B1-derived sequence of A and theCYP1B1-derived sequence of B, in the polypeptide of the invention.Typically, the amino acid will be one of the twenty naturally occurringamino acids, e.g., Ala, Leu, Ile, or Gly, and in general can be anyamino acid except (1) the one that naturally flanks the carboxy terminusof A in CYP1B1, and (2) the one that naturally flanks the amino terminusof B in CYP1B1.

[0021] By “spacer sequence” is meant a sequence of two or more aminoacids inserted between two neighboring segments, e.g., “A” and “B”, in apolypeptide of the invention. The sequence of the spacer is differentfrom the sequences which flank the carboxy terminus of A and the aminoterminus of B in the full length CYP1B1 protein from which A and B werederived. Thus, the spacer sequence forms a point of discontinuity fromboth the CYP1B1-derived sequence of A and the Y-derived sequence of B inthe polypeptide of the invention.

[0022] Examples of spacer sequences include Ala Ala, Ala Leu, Leu Leu,Leu Ala, Leu Ile, Ala Ala Ala, Ala Gly Leu, Phe Ile Ile, etc. Generally,the spacer sequence will include nonpolar amino acids, though polarresidues such as Glu, Gln, Ser, His, and Asn could also be present,particularly for spacer sequences longer than three residues. The onlyouter limit on the total length and nature of each spacer sequencederives from considerations of ease of synthesis, proteolyticprocessing, and manipulation of the polypeptide and/or nucleic acid. Itis generally unnecessary and probably undesirable to use spacersequences longer than about four or five residues, though they could be,for example, up to 6, 8, 10, 15, 20, 30, or 50 residues. Of course, theycould be even longer than 50 residues.

[0023] Spacer amino acids and spacer sequences are useful for alteringprotein stability or for promoting processing to release epitopes. Thespacers are typically removed from the polypeptide by proteolyticprocessing in the cell, along with any sequence between epitopes withina given segment. This leaves the epitopes intact for binding to MHCmolecules or (upon secretion from the cell) antibodies. Occasionally aspacer amino acid or part of a spacer sequence will remain attached toan epitope through incomplete processing. This generally will havelittle or no effect on binding to the MHC molecule.

[0024] The hybrid polypeptide encoded by a nucleic acid described hereincan further include additional segments of CYP1B1, e.g. a third, fourth,fifth, or sixth segment. These additional segments are each at leasteight amino acids in length and constitute non-contiguous portions ofCYP1B1.

[0025] The spacer can optionally encode a T cell and/or B cell epitopefrom a protein other than CYP1B1. For example, the spacer can encode thetetanus toxoid or a PADRE T cell epitope (see, e.g., U.S. Pat. No.5,662,907).

[0026] The invention also features a composition containing a nucleicacid described herein and an adjuvant or immunostimulatory agent.Adjuvants and “immunostimulatory agents” refer to substances thatstimulate an immune response in a non-antigen specific manner or inducedifferentiation or activation of professional antigen presenting cellssuch as dendritic cells. Examples of adjuvants include alum, gold,monophosphoryl lipid A, saponin, oil based emulsions, QS21, and Freund'sadjuvant. Examples of immunostimulatory agents include: a CpG containingoligonucleotide of, e.g., 18-30 nucleotides in length; cytokines such asIL-12, GM-CSF, IL-2, or IFN-gamma; cell surface receptors such as B7-1,B7-2, CCR5; and lipids, nucleic acids, carbohydrates, and bacterialpolypeptides.

[0027] The invention also includes a composition containing a nucleicacid described herein and a nucleic acid encoding an immunostimulatoryagent, e.g., IL-12, GM-CSF, IL-2, IFN-gamma, or a bacterial polypeptide.

[0028] The invention also includes a therapeutic composition containinga nucleic acid described herein and a pharmaceutically acceptablecarrier. The invention also includes a microparticle, e.g., amicrosphere containing a polymeric matrix or shell and a nucleic aciddescribed herein. The invention also includes a polymeric network, e.g.,a hydrogel and a nucleic acid described herein.

[0029] In another aspect, the invention features a method of inducing animmune response in a mammal by administering a nucleic acid describedherein to the mammal. In one example, the mammal suffers from or is atrisk for cancer. The nucleic acid can be administered by various routes,e.g., subcutaneously, intranasally, or intramuscularly. Injection of thenucleic acid may be followed by electroporation at the injection site.The immune response generated by this method can be directed to CYP1B1.The method can generate a T cell response and/or a B cell response.

[0030] The invention also features a method of inducing an immuneresponse in a mammal by administering a microparticle or polymericnetwork described herein to the mammal.

[0031] The invention also features a method of generating an immuneresponse that includes the steps of: (1) detecting a tumor or expressionof CYP1B1 in a tumor of a mammal; and (2) administering a nucleic aciddescribed herein to the mammal, wherein the administration results inthe generation of an anti-CYP1B1 immune response in the mammal.

[0032] The invention also features a method of reducing tumor growth ortumor activity in a mammal. The method includes the following steps: (1)identifying a mammal having a tumor; (2) administering a nucleic aciddescribed herein to the mammal; and (3) detecting a reduction in thesize or activity of the tumor following the administration of thenucleic acid. As used herein, “tumor activity” refers to soluble factorssecreted by a tumor cell that promote tumor cell growth. The method canfurther include a step of detecting CYP1B1 expression in the tumorbefore administering the nucleic acid.

[0033] The invention also features a method of increasing the time torelapse, life expectancy, or quality of life of a mammal. The methodincludes the following steps: (1) identifying a mammal having a tumor;(2) administering a nucleic acid described herein to the mammal; and (3)measuring an increase in the time to relapse, life expectancy, orquality of life of the mammal following administration of the nucleicacid. Increases in time to relapse, life expectancy, or quality of lifecan be measured, e.g., by a decreased need for chemotherapy or adecrease in duration of chemotherapy administration, a decreased needfor pain medication or a decrease in duration of pain medicationadministration, or a decreased need or duration of hospitalization ormedical treatment.

[0034] In another aspect, the invention features a method of inducing animmune response by delivering a nucleic acid described herein to a cell.The induction of the immune response can occur in vitro, in vivo, or exvivo. For example, anti-CYP1B1 T cells can be generated in cell culture,e.g., by incubation with antigen presenting cells such as dendriticcells expressing CYP1B1 or pulsed with CYP1B1 protein or peptides, andthen reintroduced into an individual, e.g., an individual suffering fromor at risk of having cancer.

[0035] In another aspect, the invention features a method of inducing animmune response in a mammal by administering a nucleic acid to themammal. In this method the mammal belongs to a first species, e.g., themammal is a human, and the nucleic acid encodes a polypeptide thatcontains CYP1B1 or portion thereof that binds to an MHC class I or classII molecule or immunoglobulin receptor, wherein the CYP1B1 or portionthereof is identical to a sequence of a naturally occurring CYP1B1polypeptide of a second species, e.g., a rodent such as a rat or amouse.

[0036] The nucleic acid administered according to this method can be anynucleic acid that encodes a polypeptide that contains CYP1B1 or portionthereof that binds to an MHC class I or class II molecule orimmunoglobulin receptor. In one example, the nucleic acid is a nucleicacid of the invention. The nucleic acid can be delivered to the mammalas a naked nucleic acid or associated with a delivery vehicle such as amicroparticle. Preferably the CYP1B1 or portion thereof that binds to anMHC class I or class II molecule is not identical to a sequence of anaturally occurring CYP1B1 polypeptide of the first species. The nucleicacid can optionally be administered together with an immunostimulatoryagent, as described herein.

[0037] The mammal to which the nucleic acid is administered may have orbe at risk of having a cellular proliferative disorder, e.g., cancer. Inone example, the mammal is identified as having a cellular proliferativedisorder, e.g., a tumor, prior to administering the nucleic acid to themammal.

[0038] An advantage of the invention is that the nucleic acids describedherein permit the translation of the CYP1B1 polypeptide or a portionthereof in a cell where a CYP1B1 polypeptide is either not normallyproduced or is produced at low levels.

[0039] A further advantage of some of the nucleic acid constructsdescribed herein is that they permit the translation of polypeptideswith altered stability and/or altered, reduced, or absent enzymaticactivity. Altering the stability of a polypeptide can enhance itsprocessing and subsequent recognition by the immune system. Altering theenzymatic activity of a CYP1B1 protein reduce or eliminate an unwantedbiological activity.

[0040] A further advantage of selected constructs of the invention isthat they permit the delivery of MHC class I- or class II-bindingepitopes from polypeptides having only a partial or altered sequence ofa CYP1B1 protein. Thus, deleterious effects associated with expressionof the full length CYP1B1 polypeptide are avoided. In addition,alterations in the coding sequence of the encoded CYP1B1 protein couldbreak self-tolerance to the antigen.

[0041] A further advantage of selected constructs of the invention isthat the assortment of epitopes within the polypeptides described hereinincreases the likelihood that at least one, and generally more than one,CYP1B1 epitope will be presented by each of a variety of HLA allotypes.This allows for immunization of a population of individuals polymorphicat the HLA locus, using a single nucleic acid encoding the polypeptide.

[0042] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Suitable methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0043] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIGS. 1A-1D depict the sequence of a CYP1B 1-encoding nucleicacid (SEQ ID NO:1) and polypeptide (SEQ ID NO:2).

[0045] FIGS. 2A-2C are schematic drawings of CYP1B1 cDNA constructscontaining an open reading frame (ORF) and various amounts ofuntranslated region (UTR).

[0046]FIG. 3 is a schematic drawing of a CYP1B1 polypeptide (SEQ IDNO:2) and several CYP1B1 truncation and deletion mutants.

[0047]FIG. 4 depicts the generation of CYP1B1 reactive T cells in miceimmunized with CYP1B1-expressing vectors.

[0048]FIG. 5 depicts the generation of CYP1B1 reactive T cells in miceimmunized with CYP1B1 variant-expressing vectors.

[0049]FIG. 6 depicts the generation of CYP1B1 reactive T cells in miceimmunized with CYP1B1-expressing vectors followed by electroporation.

[0050]FIG. 7 depicts the generation of CYP1B1 reactive T cells in miceimmunized with CYP1B1-expressing vectors contained in polymericnetworks.

[0051]FIG. 8 depicts the generation of CYP1B1 reactive T cells in miceimmunized with CYP1B1-expressing vectors contained in microparticles.

[0052]FIG. 9 depicts the generation of CYP1B1 reactive MHC class IIrestricted T cells in mice immunized with CYP1B1 expressing vectors.

DETAILED DESCRIPTION

[0053] The present invention provides nucleic acids containingtranscriptional units that encode CYP1B1 polypeptides or portionsthereof, wherein the transcriptional units lack sequences found in theuntranslated region (UTR) of naturally occurring forms of the CYP1B1transcript. Naturally occurring forms of a CYP1B1 transcript are thoughtto contain translational repressor elements that contribute to thepartial or total suppression of translation, at least in some cellularenvironments. The nucleic acids of the invention lack translationalrepressor elements and thus provide for a system of enhanced translationof the CYP1B1 polypeptide or portions thereof.

[0054] Nucleic acids of the invention are useful as tools for generatingor enhancing a CYP1B1-sepcific immune response in an individual. Becausethe nucleic acids lack translational repressor elements contained innaturally occurring CYP1B1 transcripts, they allow for production of aCYP1B1 protein or portion thereof and thus promote the development of animmune response. Because the nucleic acids of the invention can encodemultiple CYP1B1 epitopes, they are useful in generating immune responsesin a population containing a wide variety of MHC allotypes. The nucleicacids of the invention can also contain mutations, deletions,insertions, or rearrangements that help to promote the immunogenicity ofthe encoded protein. In this way, a protein containing an altered CYP1B1sequence may cause tolerance to self may to be broken. In addition, thenucleic acids of the invention, because they lack translationalrepressor elements contained in naturally occurring CYP1B1 transcripts,are useful for generating CYP1B1 polypeptides or portions thereof,either in vitro or in vivo.

[0055] Nucleic Acids

[0056] The nucleic acids of the invention contain a transcriptional unitthat (1) encodes a CYP1B1polypeptide or a portion thereof, but (2) doesnot correspond to a naturally occurring CYP1B1 transcript. The sequenceof a human CYP1B1 nucleic acid (SEQ ID NO:1) and protein (SEQ ID NO:2)are depicted in FIGS. 1A-1D. These sequences are used herein asreferences to describe nucleic acids of the invention. Sequences ofnaturally-occurring human CYP1B1 nucleic acids are described in GenBank™Accession U03688 and Tang et al. (1996) J. Biol. Chem. 271:28324, thecontents of which are incorporated by reference. Orthologous CYP1B1sequences have been identified in other mammals, such as rat and mouse(Walker et al. (1995) Carcinogenesis 16:1319; Shen et al. (1994) DNACell Biology 13:763; Savas et al. (1994) J. Biol. Chem. 269:14905;herein incorporated by reference). Modified forms of eukaryotic CYP1B1nucleic acids, e.g., human, rat, and mouse, are encompassed by theinvention.

[0057] The nucleic acids of the invention differ from naturallyoccurring CYP1B1 nucleic acids in that the transcriptional units lackcertain sequences contained in the 5′ and/or 3′ UTR that act astranslational repressor elements. The identity of a translationalrepressor element can be determined in various ways.

[0058] In a first method of determining whether a nucleotide sequencecontains a translational repressor element, the sequence of the fulllength RNA is analyzed, e.g., by a computer program, for the presence ofconsensus sequences associated with translational repression. Forexample, a computer analysis can identify sequences that may be bound byrepressor agents, e.g., repressor proteins. In another example, acomputer analysis can identify sequences that act as RNA destabilizationsequences, e.g., nucleotide sequences whose presence reduces the halflife of a transcript, at least in certain cellular environments. Inanother example, a computer analysis can identify consensus sequencesthat may form a secondary structure in a transcript and that areassociated with translational repression.

[0059] An analysis of the UTR of the CYP1B1 RNA transcript correspondingto the sequence of SEQ ID NO:1 identified regions of putative secondarystructure. This analysis was performed using the program mfold version3.0 by Zuker and Turner (Zuker et al. (1999) Algorithms andThermodynamics for RNA Secondary Structure Prediction: A PracticalGuide, In RNA Biochemistry and Biotechnology, 11-43, J. Barciszewski &B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers;Mathews et al. (1999) J. Mol. Biol. 288:911-940). mfold is a animplementation of the Zuker algorithm for RNA secondary structureprediction based on free energy minimization. The folding temperature inthis analysis is fixed at 37° C.

[0060] Regions of predicted secondary structure in the CYP1B1 RNAtranscript are depicted in Table 1. Because these regions constituteputative repressors of translation, a nucleic acid of the invention canlack one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 13, 14,or 15, of these sequences, SEQ ID NOs:3-17. TABLE 1 Secondary StructureAnalysis of Untranslated Regions of the CYP1B1 Transcript Location inSEQ ID NO:1 UTR Length SEQ ID NO: nucleotides 42-62 5' 21 3 nucleotides72-112 5' 41 4 nucleotides 262-322 5' 61 5 nucleotides 262-362 5' 101 6nucleotides 2022-2092 3' 71 7 nucleotides 2092-2162 3' 71 8 nucleotides2222-2242 3' 21 9 nucleotides 2092-4272 3' 2181 10 nucleotides 2287-27623' 476 11 nucleotides 2252-4972 3' 2721 12 nucleotides 4352-4812 3' 46113 nucleotides 4372-4822 3' 451 14 nucleotides 4832-4892 3' 61 15nucleotides 4942-5002 3' 61 16 nucleotides 5012-5134 3' 123 17

[0061] In a second method of determining whether a nucleotide sequencecontains a translational repressor element, translational repressorelements can be identified by an empirical determination of the amountof protein produced by a modified CYP1B1 transcript as compared to awild type CYP1B1 transcript. For example, a UTR deletion mutant of thesequence of SEQ ID NO:1 is constructed, the mutant and the sequence ofSEQ ID NO:1 are each expressed in separate cell populations, and theamount of CYP1B1 protein produced from each of the mutant and the wildtype is compared. If the modified transcript results in enhanced proteinproduction as compared to the wild type CYP1B1, then it is expected tohave one or more translational repressor elements. For example, anucleic acid of the invention may lack the 5′ UTR sequence of SEQ IDNO:18 (nucleotides 1-362 of SEQ ID NO:1). In another example, a nucleicacid can lack the 3′ UTR sequence of SEQ ID NO:19 (nucleotides 2011-5128of SEQ ID NO:1). In another example, a nucleic acid can lack both SEQ IDNO:18 and 19. In addition to nucleic acids lacking all of SEQ ID NO:18and/or SEQ ID NO:19, the invention also includes nucleic acids that lacka specific number of consecutive nucleotides, e.g., at least 400, 300,200, 150, 100, 50, 25, or 10, of SEQ ID NO:18 and/or SEQ ID NO:19. Thisanalysis can be performed in conjunction with the computer analysisdescribed above.

[0062] The nucleic acids of the invention can also differ from naturallyoccurring CYP1B1 nucleic acids in that they may contain mutations,deletions, insertions, or rearrangements that help to promote theimmunogenicity of the encoded CYP1B1 protein or variant thereof. Methodsof determining immunogenicity of a protein are well known in the art andinclude immunization of an animal with the nucleic acid, and subsequentremoval and analysis of the lymph node, spleen, blood, or serum for Tcell or B cell responses. Standard assays are described herein andinclude Cr51, Elispot, tetramer, ELISA, and intracellular cytokinestaining analysis by FACS to measure cytotoxic T cells (CTL) specificfor CYP1B1 , Elispot and T cell proliferation studies to measure Thelper responses, and ELISA and western analysis to measure B cellresponses.

[0063] Modifications to a CYP1B1 nucleic acid, e.g., SEQ ID NO:1, can bemade by methods well known to those of skill in the art. Deletions ofparticular 5′ and/or 3′ UTR sequences can be achieved by PCRamplification of a template using appropriate primer pairs andsubcloning of the amplified product into an expression vector. Forexample, a nucleic acid lacking SEQ ID NO:18 and SEQ ID NO:19 can beconstructed by PCR amplification, with the nucleic acid of SEQ ID NO:1as a template, using primers that correspond to regions 363-382 of SEQID NO:1 (primer 1) and regions 1991-2010 of SEQ ID NO:1 (primer 2).

[0064] FIGS. 2A-2C depict three examples of CYP1B1 -encoding nucleicacids. These figures refer to nucleotide positions of SEQ ID NO:1. FIG.2A is the full length CYP1B1 nucleic acid of SEQ ID NO:1. FIG. 2B is atruncated form of SEQ ID NO:1, lacking a portion of the 5′ UTR and 3′UTR. FIG. 2C lacks all 5′ UTR as well as all 3′ UTR sequences present inthe CYP1B1 nucleic acid of FIG. 2A.

[0065] Regulatory elements can be included in the nucleic acid tofacilitate expression of the nucleic acid encoding the polypeptide.These elements include sequences for enhancing expression in human orother mammalian cells, e.g., promoters and/or enhancers. For example, aT7 polymerase promoter, a viral promoter such as CMV, RSV, or LTR, atissue-specific promoter such as a muscle-specific promoter, acell-specific promoter such as an APC-specific promoter, or an induciblepromoter is optionally present at the 5′ end of the coding sequence.Examples of inducible promoters include a metallothionine promoter (see,e.g., Testa et al. (1994) Cancer Res. 54:4508) an atetracycline-responsive promoter (see, e.g., Giavazzi et al. (2001)61:309)

[0066] The nucleic acid can also include an RNA stabilization sequence,e.g., an RNA stabilization sequence derived from the Xenopus laevisβ-globin gene, 5′ and/or 3′ to the coding sequence; an intron (which canbe placed at any location within or adjacent to the coding sequence); apoly(A) addition site; an origin of replication; and one or more genesencoding selectable markers, e.g., a kanarnycin resistance gene orauxotrophic marker, enabling the constructs to replicate and be selectedin prokaryotic and/or eukaryotic hosts.

[0067] The nucleic acid can also include a translational regulatorysequence that is not derived from a naturally occurring CYP1B1transcript. Examples of translational regulatory sequence are known inthe art (see, e.g., Aziz and Munro (1987) Proc. Natl. Acad. Sci. USA84:8478). The addition of a translational regulatory sequence to atranscriptional unit described herein can allow for translationalregulation of protein expression. For example, the first 67 nucleotidesof the 5′ UTR of the ferritin mRNA (Aziz and Munro supra) can be coupledto a transcriptional unit described herein to render translation of theencoded protein an iron-responsive event.

[0068] The nucleic acid may also contain other transcriptional andtranslational signals, such as a Kozak sequence, as well as a sequenceencoding an antibody determinant such as a FLAG, myc, HA, or His tag,optionally present at the 5′ or 3′ end of the coding sequence before thetermination codon.

[0069] Nucleic acids encoding CYP1B1 polypeptides can be used in anyvector that allows for expression in antigen-presenting cells (APC) of amammal. The nucleic acid may be cloned into an expression vector, i.e.,a vector in which the coding sequence is operably linked to expressioncontrol sequences. Vectors useful in this invention include linearnucleic acid fragments or circular DNAs, plasmid vectors, viral vectors,fungal vectors, and bacterial vectors. A “plasmid” is an autonomous,self-replicating, extrachromosomal, circular DNA. Preferred viralvectors are those derived from retroviruses, adenovirus,adeno-associated virus, pox viruses, SV40 virus, alpha viruses or herpesviruses. An example of a suitable vector is the family of pcDNAmammalian expression vectors (Invitrogen).

[0070] A nucleic acid can encode a single polypeptide or multiplepolypeptides, each under the control of a different promoter, e.g., dualpromoter vectors. A dual promoter vector permits two shorterpolypeptides to replace the single longer version, with no loss in thenumber of epitopes produced from a given vector. It also allows addingnew CYP1B1 sequences without altering the sequence, and perhaps theprocessing, of the first polypeptide. It also allows coding of twounrelated proteins such as CYP1B1 and an immunostimulating agent.Alternatively, a nucleic acid contains IRES sequences located betweentwo coding sequences, e.g., between nucleic acid sequences encoding twopolypeptides described herein. The IRES sequences cause the ribosome toattach to the initiator codon of the downstream translational unit andtranslate a second protein from a single polycistronic mRNA. Expressionvectors encoding two or more polypeptides can optionally encode onesecreted polypeptide and one non-secreted polypeptide. Such a vector canbe used to induce both a T cell response and a B cell response. It alsocan be used to code for two unrelated proteins such as CYP1B1 and animmunostimulating agent.

[0071] CYP1B1 Polypeptides

[0072] The nucleic acids of the invention encode polypeptides containingCYP1B1 or a portion thereof that contains at least one peptide epitopethat binds to an MHC class I or class II molecule or immunoglobulinreceptor. The nucleic acids encoding the polypeptide described hereincan encode a methionine residue at the amino terminus of the polypeptideto facilitate translation. The polypeptide can contain multiple epitopesof CYP1B1 as well as multiple segments of CYP1B1, each of which containsone or more epitopes. MHC-binding epitopes of CYP1B1 can be identifiedby methods well known to those of skill in the art. MHC class I-bindingpeptides are typically 8-10 amino acid residues in length, whereas MHCclass II-binding peptides are typically 12-30 amino acid residues inlength.

[0073] Epitopes that bind to a specific MHC allele can be identified byfirst synthesizing a series of overlapping peptide fragments from CYP1B1and testing the peptides in art-recognized binding studies with a radiolabeled peptide known to bind to the MHC allele. If a test peptidedemonstrates specific binding to an MHC allele as measured by, forexample, competition with the radio labeled test peptide (i.e., it is anepitope), the epitope can be combined with additional epitopes(overlapping or adjacent) to produce or define a segment. Examples ofthese and related methods can be found in U.S. Pat. No. 6,037,135, WO99/45954, and WO 044775A2, herein incorporated by reference.

[0074] Alternatively, epitopes can be identified by refolding solubleMHC class I molecules in the presence of radiolabeled β2-microglobulinand a test peptide. The complete complex will refold and produce areceptor of the correct size. β2-microglobulin dissociates from thecomplex at a measurable rate that is directly correlated with thebinding affinity of the test peptide (Garboczi et al. (1992) Proc. Nat.Acad. Sci. USA 89:3429; Parker et al. (1992) J. Biol. Chem. 267:5451;and Parker et al. (1992) J. Immunol. 149:1896). Analysis of this type ofdata has resulted in an algorithm that predicts the dissociation timesof a given test peptide for an HLA-A2 receptor (Parker et al. (1994) J.Immunol. 152:163). Fast dissociation has been correlated with lowaffinity, and slow dissociation with high affinity. This algorithm hasbeen expanded and is available for predicting binding affinity ofepitopes for the HLA-A allotypes, -A1, -A2, -A3, -A11, and -A24. Thealgorithm can be found at the web site(http://wwwbimas.dcrt.nih.gov/molbio/hla_bind/index.html). For anepitope to generate effective cytotoxic T cell (CTL) responses, it mustbind to an MHC molecule on an antigen-presenting cell (APC), and theresulting receptor-ligand complex must be recognized by a T cellreceptor expressed on the CTL.

[0075] Alternatively, epitopes may be identified by identifying MHCclass I or class II-binding peptides using techniques described in,e.g., U.S. Pat. No. 5,827,516, and U.S. Ser. No. 09/372,380, hereinincorporated by reference.

[0076] Epitopes that bind in vitro to MHC molecules as described abovecan be analyzed for their effectiveness at stimulating human Tcell-responses (or used to generate a T cell response) in an in vitroimmunization assay (see, e.g., Schultze et.al. (1997) J. Clin. Invest.100:2757). Such an assay has been used previously to identify human andmurine T cell-responsive epitopes (Alexander et al. (1996) Amer. J.Obstet. and Gynecol. 175:1586; Tarpey et al. (1994) Immunology 81:222).These assays have also been used to generate large numbers of specificCTL for immunotherapy (Tsai et al. (1998) Crit. Rev. Immunol. 18:65). Toensure reliability, it is desirable to perform the first round of T cellstimulation in the presence of dendritic cells (DCs) pulsed with thetest peptide. Moreover, inclusion of IL-10 during the stimulation maysuppress the non-specific responses that may sometimes arise duringculture of the cells. T cell activation may then be examined using anELISA assay to measure λ-IFN secretion, by ELISPOT to measure cytokinessuch as IL-10, IL-4, TNFα, IFN-γ or IL-2, or by use of FACS to determinethe increase in CD8+, CD16- cells containing cytokines such as λ-IFN bytricolor analysis. Alternatively, T cell activation can be measuredusing a ⁵¹Cr release CTL assay or a tetramer-based assay (see, e.g.,Molldrem et.al. (2000) Nature Med. 6:1018).

[0077] It is possible that not every individual with a given allotypewill respond to a particular epitope. For example, one individual whosecells bear the HLA-A2 allotype may respond to a given epitope, whereas asecond such individual may not. To overcome this difficulty, T cellsfrom two donors, and even more preferably three donors, for each HLAallotype can be tested to verify that it is a T cell epitope. For themore common alleles (i.e., HLA-A2 and -A3) up to four donors arepreferably tested.

[0078] Each epitope is tested initially with cells from one donor. If anepitope does not stimulate a T cell response using cells of the firstdonor, it is tested again with cells from a second donor, and then athird donor. If the epitope does not demonstrate T cell reactivity aftertwo or three attempts, it is optionally not chosen for inclusion in apolypeptide.

[0079] Altering the method by which the in vitro presentation of antigenis performed may enhance analysis. An initial stimulation of T cellswith DCs is typically part of the in vitro immunization. To enhance theimmunization, DCs can be added at each round of stimulation to ensureadequate antigen presentation and T cell stimulation, e.g., usingpreviously generated and subsequently frozen DCs. Alternatively,enhanced T cell stimulation can be achieved by activating the antigenpresenting cells (APC) with an antibody binding to the APCs cell surfaceCD40 receptor.

[0080] Alternatively, the epitopes can be selected for inclusion in theconstruct based solely on their binding affinity to HLA molecules, oridentified based upon analysis of naturally processed peptides, asdescribed herein and in Chicz et al. (1993) J. Exp. Med. 178:27 and U.S.Pat. No. 5,827,516.

[0081] B cell epitopes can be selected based on their ability to induceimmune responses in mammals. For example, CYP1B1 peptides are mixed withfreund's adjuvant and injected into mice. Serum from immunized animalsis collected and tested by Western blot for reactivity to CYP1B1(commercially available, Gentest, Woburn Mass.).

[0082] Polypeptides encoded by nucleic acids of the invention do notnecessarily include the full length CYP1B1 protein of SEQ ID NO:2. Forexample, a polypeptide encoded by a nucleic acid of the invention canlack the bioactivation properties of naturally occurring CYP1B1 (see,e.g., Heidel et al. (2000) Cancer Res. 60:3454). A nucleic acid encodinga CYP1B1 polypeptide or portion thereof can include a coding sequencethat contains a loss of function mutation, e.g., an insertion, adeletion, a frame shift mutation, or a single nucleotide mutation(Bailey et al. (1998) Cancer Res. 58:5038). Examples of frame shiftmutations in a CYP1B1 coding sequence are described in Stoilov et al.(1997) Hum. Mol. Gen. 6:641 and Sarfarazi et al. (1997) Hum. Mol. Gen.6:1667. In another example, a polypeptide can lack all or part of thehinge region (e.g., about amino acids 38-61 of SEQ ID NO:2) or conservedcore sequence of the heme-binding portion of CYP1B1 located betweenabout amino acids 400-540 of SEQ ID NO:2 ( Stoilov et al. (1998) Am. J.Human Genet. 62:573). In another example, a polypeptide can lack all orpart the oxidation-reduction domain of CYP1B1 , or have mutations inactive site regions (Lewis et al. (1999) Toxicology 139:53), or inregions required for protein folding or stability. In another example, apolypeptide can lack all or part of a CYP1B1 transmembrane regionlocated between about amino acids 18-37 of SEQ ID NO:2.

[0083]FIG. 3 depicts a CYP1B1 polypeptide (SEQ ID NO:2) and fragmentsand variants thereof. The various polypeptides depicted in FIG. 3 can beencoded by CYP1B1-encoding nucleic acids described herein, e.g., nucleicacids lacking all or a portion of a 5′ UTR and/or a 3′ UTR. Some of thepolypeptides include the peptide of SEQ ID NO:22. The following is adescription of the CYP1B1 fragments and variants depicted in FIG. 3.

[0084] SEQ ID NO:31

[0085] This polypeptide contains a deletion of four amino acids(residues 51-54 of SEQ ID NO:2) in the hinge region of CYP1B1 . Thispolypeptide contains amino acid changes, as compared to the wild typeprotein, at residues 57 (W to C), 61 (G to E), 365 (G to W), 379 (P toL), and 387 (E to K). The altered residues are underlined. The aminoacid sequence of SEQ ID NO:31 is recited as follows:MGTSLSPNDPWPLNPLSIQQTTLLLLLSVLATVHVGQRLLRQRRRQLRSAFACPLIENAAAVGQAAHLSFARLARRYGDVFQIRLGSCPIVVLNGERAIHQALVQQGSAFADRPAFASFRVVSGGRSMAFGHYSEHWKVQRRAAHSMMRNFFTRQPRSRQVLEGHVLSEARELVALLVRGSADGAFLDPRPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTVFREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMIFSVGKRRCIGEELSKMQLFLFISILAHQCDFRANPNEPAKMNFSYGLTIKPKSFKVNVTLRESMELLDSAVQNLQAKETCQEQKLISEED L.

[0086] SEQ ID NO:32

[0087] This polypeptide contains a deletion of amino acids 1-60 of SEQID NO:2. The deletion encompasses the ER domain, transmembrane domain,and hinge region of CYP1B1. A methionine (underlined) is positioned atthe amino terminus of the polypeptide of SEQ ID NO:32. This polypeptidecontains amino acid changes, as compared to the wild type protein, atresidues 365 (G to W), 379 (P to L), and 387 (E to K). The alteredresidues are also underlined. This sequence has been denoted F1R1. Theamino acid sequence of SEQ ID NO:32 is recited as follows:MGNAAAVGQAAHLSFARLARRYGDVFQIRLGSCPIVVLNGERAIHQALVQQGSAFADRPAFASFRVVSGGRSMAFGHYSEHWKVQRRAAHSMMRNFFTRQPRSRQVLEGHVLSEARELVALLVRGSADGAFLDPRPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTVFREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMIFSVGKRRCIGEELSKMQLFLFISILAHQCDFRANPNEPAKMNFSYGLTIKPKSFKVNVTLRESMELLDSAVQNLQAKETCQEQKLISEEDL.

[0088] SEQ ID NO:33

[0089] This polypeptide contains a deletion of amino acids 1-60 and462-543 of SEQ ID NO:2. The deletion encompasses the ER domain,transmembrane domain, and hinge region of CYP1B1 . A methionine(underlined) is placed at the amino terminus of the polypeptide of SEQID NO:33. This polypeptide contains amino acid changes, as compared tothe wild type protein, at residues 365 (G to W), 379 (P to L), and 387(E to K). The altered residues are also underlined. This sequence hasbeen denoted F1R2. The amino acid sequence of SEQ ID NO:33 is recited asfollows: MGNAAAVGQAAHLSFARLARRYGDVFQIRLGSCPIVVLNGERAIHQALVQQGSAFADRPAFASFRVVSGGRSMAFGHYSEHWKVQRRAAHSMMRNFFTRQPRSRQVLEGHVLSEARELVALLVRGSADGAFLDPRPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTVFREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLI NKDLTSRVMEQKLISEEDL.

[0090] SEQ ID NO:34

[0091] This polypeptide contains a deletion of amino acids 1-171 of SEQID NO:2. The deletion encompasses the ER domain, transmembrane domain,and hinge region of CYP1B1. A methionine (underlined) is placed at theamino terminus of the polypeptide of SEQ ID NO:34. This polypeptidecontains amino acid changes, as compared to the wild type protein, atresidues 365 (G to W), 379 (P to L), and 387 (E to K). The alteredresidues are also underlined. This sequence has been denoted F2R1. Theamino acid sequence of SEQ ID NO:34 is recited as follows:MSEARELVALLVRGSADGAFLDPRPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTVFREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMIFSVGKRRCIGEELSKMQLFLFISILAHQCDFRANPNEPAKMNFSYGLTIKPKSFKVNVTLRESMELLDSAVQNLQAKETCQEQKLISEEDL.

[0092] SEQ ID NO:35

[0093] This polypeptide contains a deletion of amino acids 1-171 and462-543 of SEQ ID NO:2. The deletion encompasses the ER domain,transmembrane domain, and hinge region of CYP1B1. A methionine(underlined) is placed at the amino terminus of the polypeptide of SEQID NO:35. This polypeptide contains amino acid changes, as compared tothe wild type protein, at residues 365(G to W), 379(P to L), and 387(Eto K). The altered residues are also underlined. This sequence has beendenoted F2R2. The amino acid sequence of SEQ ID NO:35 is recited asfollows: MSEARELVALLVRGSADGAFLDPRPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTVFREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMEQKLISEED L.

[0094] SEQ ID NO:36

[0095] This polypeptide contains a deletion of amino acids 1-292 of SEQID NO:2. The deletion encompasses the ER domain, transmembrane domain,and hinge region of CYP1B1. This polypeptide contains amino acidchanges, as compared to the wild type protein, at residues 365 (G to W),379 (P to L), and 387 (E to K). The altered residues are underlined.This sequence has been denoted F3R1. The amino acid sequence of SEQ IDNO:36 is recited as follows:MDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMIFSVGKRRCIGEELSKMQLFLFISILAHQCDFRANPNEPAKMNFSYGLTIKPKSFKVNVTLRESMELLDSAVQNLQAKETC QEQKLISEEDL.

[0096] SEQ ID NO:37

[0097] This polypeptide contains a deletion of amino acids 1-292 and462-543 of SEQ ID NO:2. The deletion encompasses the ER domain,transmembrane domain, and hinge region of CYP1B1 . This polypeptidecontains amino acid changes, as compared to the wild type protein, atresidues 365 (G to W), 379 (P to L), and 387 (E to K). The alteredresidues are underlined. This sequence has been denoted F3R2. The aminoacid sequence of SEQ ID NO:37 is recited as follows:MDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMEQKLISEEDL.

[0098] An insertion, deletion, frame shift mutation, or singlenucleotide mutation can be introduced into a CYP1B1 polypeptide sequenceto result in a polypeptide with an altered stability and/or biologicalactivity as compared to the wild type protein. By altering the stabilityof a CYP1B1 polypeptide, this can affect its processing by the cellularmachinery. For example, a CYP1B1 polypeptide with decreased stability isexpected to undergo increased processing, thereby resulting in anincrease in CYP1B1 peptides presented by MHC class I and/or class IImolecules. By altering (e.g., reducing or eliminating) the biologicalactivity of CYP1B1 , unwanted activity such as enzymatic activity can bereduced or eliminated.

[0099] The following are examples of CYP1B1 polypeptides containingalterations at three (SEQ ID NO:38) and five (SEQ ID NO:39) amino acidresidues, as compared to the wild type CYP1B1 protein.

[0100] SEQ ID NO:38

[0101] This polypeptide contains amino acid changes, as compared to thewild type protein, at residues 57 (W to C), 61 (G to E), and 365 (G toW). The altered residues are underlined. The amino acid sequence of SEQID NO:38 is recited as follows:MGTSLSPNDPWPLNPLSIQQTTLLLLLSVLATVHVGQRLLRQRRRQLRSAPPGPFACPLIENAAAVGQAAHLSFARLARRYGDVFQIRLGSCPIVVLNGERAIHQALVQQGSAFADRPAFASFRVVSGGRSMAFGHYSEHWKVQRRAAHSMMRNFFTRQPRSRQVLEGHVLSEARELVALLVRGSADGAFLDPRPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTVFREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLPYVLAFLYEAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMIFSVGKRRCIGEELSKMQLFLFISILAHQCDFRANPNEPAKMNFSYGLTIKPKSFKVNVTLRESMELLDSAVQNLQAKETCQ.

[0102] SEQ ID NO:39

[0103] This polypeptide contains amino acid changes, as compared to thewild type protein, at residues 57 (W to C), 61 (G to E), 365 (G to W),379 (P to L), and 387(E to K). The altered residues are underlined. Theamino acid sequence of SEQ ID NO:39 is recited as follows:MGTSLSPNDPWPLNPLSIQQTTLLLLLSVLATVHVGQRLLRQRRRQLRSAPPGPFACPLIENAAAVGQAAHLSFARLARRYGDVFQIRLGSCPIVVLNGERAIHQALVQQGSAFADRPAFASFRVVSGGRSMAFGHYSEHWKVQRRAAHSMMRNFFTRQPRSRQVLEGHVLSEARELVALLVRGSADGAFLDPRPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTVFREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAEKKAAGDSHGGGARLDLENVPATITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVWRDRLPCMGDQPNLLYVLAFLYKAMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPVKWPNPENFDPARFLDKDGLINKDLTSRVMIFSVGKRRCIGEELSKMQLFLFISILAHQCDFRANPNEPAKMNFSYGLTIKPKSFKVNVTLRESMELLDSAVQNLQAKETCQ.

[0104] As described above, polypeptides encoded by the nucleic acidsdescribed herein include all of CYP1B1 or a portion thereof that bindsto an MHC class I or class II molecule or immunoglobulin receptor. Apolypeptide can contain 25, 50, 150, 200, 250, 300, 400, 500 or moreamino acids corresponding to a sequence of consecutive amino acidspresent in SEQ ID NO:2. Additionally, the polypeptide can be less than300, 200, 150, 100, or 50 amino acids in length. For example, thepolypeptide can contain SEQ ID NO:20 (amino acids 1-272 of SEQ ID NO:2)or SEQ ID NO:21 (amino acids 273-544 of SEQ ID NO:2).

[0105] Any of the CYP1B1 polypeptides or fragments thereof describedherein can contain all or a portion of a sequence identical to the wildtype CYP1B1 protein or an altered CYP1B1 sequence. For example,polypeptides having the structure of any of those described herein(e.g., the polypeptides depicted in FIG. 3) can be made so as to includeany one or more (e.g., one, two, three, four, or five) of the amino acidalterations contained in the polypeptides of SEQ ID NO:38 and/or SEQ IDNO:39.

[0106] A polypeptide encoded by a nucleic acid described herein cancontain the amino acid sequence FLDPRPLTV (SEQ ID NO:22), whichcorresponds to amino acid residues 190-198 of SEQ ID NO:2). The peptideof SEQ ID NO:22 is a naturally processed epitope of the CYP1B1polypeptide. Additionally, a polypeptide can include at least 8 aminoacids derived from the sequence of SEQ ID NO:23 (amino acid residues185-205 of SEQ ID NO:2). A polypeptide containing SEQ ID NO:22 or SEQ IDNO:23 can be less than 300, 200, 150, 100, or 50 amino acids in length.

[0107] The nucleic acids of the invention may in addition include one ormore sequences encoding targeting signals that direct the polypeptide toa desired intracellular compartment, the targeting signal being linkedto the polypeptide. Targeting signals (the term is used interchangeablywith trafficking signal or targeting sequence) can target the proteinfor secretion or can direct the polypeptide to endoplasmic reticulum(ER), the golgi, the nucleus, a lysosome, a class II peptide loadingcompartment, or an endosome, and include signal peptides (the aminoterminal sequences which direct proteins into the ER duringtranslation), ER retention peptides such as KDEL (SEQ ID NO:24), andlysosome-targeting peptides such as KFERQ (SEQ ID NO:25), QREFK (SEQ IDNO:26), and other pentapeptides having Q flanked on one side by fourresidues selected from K, R, D, E, F, I, V, and L. Also included aretargeting signals that direct the secretion of the polypeptide (e.g.,SEQ ID NO:40). Also included are targeting signals that direct insertionof the polypeptide into a membrane (e.g., a transmembrane sequence).Polypeptides including a membrane insertion sequence can be constructedeither with or without a cytoplasmic tail.

[0108] An example of an ER-targeting sequence is the HLA-DRα leadersequence, MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:27). The targetingsequence may include only a portion (e.g., at least ten amino acidresidues) of this specified 25 residue sequence, provided that theportion is sufficient to cause targeting of the polypeptide to the ER.Another example of a targeting sequence is the E1A sequence.

[0109] Nuclear localization sequences include nucleoplasmin- andSV40-like nuclear targeting signals, as described in Chelsky et al.(1989) Mol. Cell Biol. 9:2487; Robbins (1991) Cell 64:615; and Dingwallet al. (1991) TIBS 16:478. Some nuclear localization sequences includeAVKRPAATKKAGQAKKK (SEQ ID NO:28), RPAATKKAGQAKKKKLD (SEQ ID NO:29), andAVKRPAATKKAGQAKKKLD (SEQ ID NO:30).

[0110] In some cases it is desirable to modify the amino acid sequenceof the targeting signal to facilitate cleavage by a signal peptidase orother proteolytic agent. Recognition sequences for signal peptidases aredescribed in Von Heijne (1986) Nucleic Acids Research 14:4683. The −3,−1 rules of von Heijne can be used to select a sequence that increasesthe probability of successful cleavage by signal peptidase when thetargeting signal is present.

[0111] In some cases it is desirable to modify the polypeptide sequence,with respect to the wild type CYP1B1 sequence, by altering the stabilityof the polypeptide. One method of decreasing the stability of apolypeptide is by facilitating its targeting it to the proteasome. Forexample, a targeting signal comprising ubiquitin sequences can be linkedto a polypeptide described herein, so as to target the polypeptide tothe cellular proteasome for degradation (see, e.g., Hochstrasser (1995)Curr. Opin. Cell. Biol. 7:215-223; Rodriguez et al. (1997) J. Virol.71:8497-8503). In another method of decreasing the stability of apolypeptide, a polypeptide can lack all or a portion of a CYP1B1sequence that contributes to protein stability, e.g., all or a portionof the hinge region (about amino acids 38-61 of SEQ ID NO:2). In oneexample, a polypeptide lacks the PPGP region located between amino acids51-54 of SEQ ID NO:2 (see, e.g., the polypeptide of SEQ ID NO:31).

[0112] Alternatively, a polypeptide can lack a targeting signal, whichwill cause the polypeptide to be located in the cytoplasm.

[0113] Once expressed in a cell, the encoded peptide can be processedinto one of several MHC class I binding epitopes. The MHC molecule, uponbinding to the peptide, can activate a T cell response. MHC class IIbinding peptides may also be generated from the encoded peptide. Thesepeptides would be expected to activate T helper cells or CTL uponpresentation by the MHC class II expressing cells. Other receptors mayalso bind the encoded peptide or its processed fragments to activateimmune cells such as NK or B cells. These cells may also be activated bycytokines elicited in response to the peptides of the invention. In oneexample, secretion signals such as SEQ ID NO:40 are added to thepolypeptide, resulting in the secretion of the polypeptide and theactivation of immune cells such as B cells.

[0114] In those polypeptides containing multiple segments of CYP1B1, theorder of the segments within the polyepitope polypeptide can correspondto the order in which the segments appear in the native CYP1B1 protein,though some of the amino acid sequence (i.e., at least one residue)between the individual segments in the native protein may be deleted.Alternatively, the segment order may differ from that in the naturallyoccurring protein. A protein created by this process may have beendesigned via molecular evolution (see, e.g., U.S. Pat. No. 6,132,970),exon shuffling or domain shuffling approaches.

[0115] To determine whether the polypeptide is processed and theepitopes are presented by MHC, an in vitro T cell stimulation assay canbe performed using autologous spleen cells, PBL, or EBV-transformedcells infected with a recombinant vaccinia virus that contain thepolyepitope polypeptide coding sequence. These target cells aregenerated by incubating spleen cells or PBL with the recombinantvaccinia at an multiplicity of infection (moi) of 3-10 plaque formingunits (pfu)/cell at 37° C. for 2 hours. After infection, cells arepelleted, washed and used as targets in the in vitro stimulation assay.The stimulated T cells from one or more individuals, e.g., a mouse or ahuman, with the different MHC allotypes (or from one or more individualsimmunized with CYP1B1 polypeptides or nucleic acid constructs) areincubated with the target cells, and the ability of the target cells tostimulate the T cells is measured, e.g., by λ-interferon expression orsecretion.

[0116] Alternatively, epitope processing from the polyepitopepolypeptide can be examined using proteasomes purified from human cells(Theobald et al. (1998) J. Exp. Med. 188:1017; Kisselev et al. (1999) J.Biol. Chem. 289:3363; and Nussbaum et al. (1998) Proc. Nat. Acad. Sci.(USA) 95:12404).

[0117] In addition to the T cell assays, an assay that utilizes mice,e.g., transgenic mice, can be used to verify that the constructfunctions (e.g., epitopes are correctly processed and presented) whendelivered in vivo. For measuring HLA-A2-restricted presentation, thepolyepitope construct in a mammalian expression vector (e.g., a plasmid)is encapsulated in microparticles and introduced into HLA-A2 transgenicmice by a route such as intramuscular or subcutaneous injection. Theconstruct may alternatively be administered without the microparticledelivery vehicle, e.g., in a recombinant viral or bacterial vector,e.g., vaccinia virus or as naked DNA. T cell responses are subsequentlyexamined in vitro (Hedley et al., Nature Med. 4:365-68, 1998). Targetcells can be T2A2 cells (T2 cells transfected with DNA encoding HLA-A2)or EL4.A2 cells (EL4 cells transfected with DNA encoding HLA-A2) pulsedwith the A2 epitope being tested and T2A2 cells into which has beenintroduced a nucleic acid of the invention. Parallel studies areperformed using EL4.A2 or T2A2 cells pulsed with no peptide or with anirrelevant peptide. In this way, HLA-A2 epitopes that are processed andpresented in vivo following administration of the nucleic acid of theinvention are identified. A positive result suggests that processing ofthe polyepitope polypeptide is occurring as predicted.

[0118] Alternately, polyepitope polypeptides may be made according tomethods of Hanke, et al. (1998) Vaccine 16:426, or as described in U.S.Ser. No. 60/154,665 and U.S. Ser. No. 60/169,846, which are herebyincorporated by reference.

[0119] Immunostimulatory Agent

[0120] A composition can include a nucleic acid as described herein aswell as an adjuvant or immunostimulatorv agent or a nucleic acidencoding an immunostimulatory agent. Examples of useful adjuvants andimmunostimulatory agents include: ISCOMS, virus like particles (VLPs),alum, gold, freund's adjuvant, cytokines such as IL-12, GM-CSF, IL-2, orIFN-gamma; cell surface receptors such as B7-1, B7-2 or CCR5;lipopolysaccharide (LPS); monophosphoryl lipid A; QS21; CpG-containingoligonucleotides, e.g., of 18-30 nucleotides in length, and bacterialpolypeptides such as a bacteriotoxin. Any compound that stimulatesdifferentiation or activation of professional antigen presenting cellssuch as dendritic cells is a useful immunostiumulatory agent. Examplesof CpG-containing oligonucleotides are described in U.S. Pat. No.6,239,116. A nucleic acid encoding a polypeptide described herein and animmunostimulatory agent can optionally be included in a single vector,e.g., a two promoter vector or IRES vector as described herein.Alternatively, a nucleic acid of the invention can encode a CYP1B1polypeptide or portion thereof fused in frame to an immunostimulatoryagent. Methods of creating such fusion proteins are well known in theart and are described in, for example, WO 95/05849.

[0121] Methods

[0122] The nucleic acids of the invention can be used as immunogens inindividuals known to have various types of cell proliferative disorders,such as lymphoproliferative disorders or cancer, individuals suspectedof having various types of cancer, or individuals susceptible to varioustypes of cancer (e.g., individuals having genetic and/or hereditaryindicia of cancer susceptibility, e.g., mutations in the BRCA1 gene).Other suitable individuals include those displaying symptoms of, orlikely to develop, cancer-associated conditions. The nucleic acids canbe used, prophylactically or therapeutically, to prevent or treatconditions associated with several different cell proliferativedisorders or cancers, e.g., cancers of the bladder, breast, colon,connective tissue, lung, esophagus, skin, lymph node, brain, ovary,stomach, uterus, testis, and prostate. In one example, the nucleic acidis used as a vaccine.

[0123] The nucleic acids encoding the peptides can administered alone orin combination with other therapies known in the art, e.g.,chemotherapeutic regimens, radiation, and surgery, to treat varioustypes of proliferative disorders or cancer, or diseases associated withthese proliferative disorders or cancers. In addition, the nucleic acidof the invention can be administered in combination with othertreatments designed to enhance immune responses, e.g., byco-administration with adjuvants, vitamins, immunostimulatory agents, orcytokines (or nucleic acids encoding cytokines), as is well known in theart. Compositions containing nucleic acids and immunostimulatory agentsare described herein.

[0124] The nucleic acid of the invention can also be used in manufactureof a medicament for the prevention or treatment of various cancers, orconditions associated with these cancers.

[0125] Nucleic acids encoding CYP1B1 polypeptides or portions thereofcan be used in immunotherapy to stimulate the immune reaction of acancer patient against a rapidly proliferating cell population or tumor,e.g., a tumor that expresses the CYP1B1 protein and presents CYP1B1peptides in the context of an MHC molecule or a tumor that expresses theCYP1B1 protein on the cell surface. Because naturally occurring forms ofa CYP1B1 transcript are thought to contain translational repressorelements that contribute to the partial or total suppression oftranslation, the immune system of an individual may be naive or tolerantto CYP1B1. CYP1B1 protein expression in other cells may result inimmunologic self-tolerance and thus mechanisms to break self-tolerancemay increase the efficacy of the nucleic acids of the invention. Forexample, the nucleic acids of the invention lack at least onetranslational repressor element and thus provide for a system ofenhanced translation of the CYP1B1 polypeptide or portions thereof,thereby enabling an individual's immune system to generate ananti-CYP1B1 immune response. A polypeptide described herein can beproduced in one cell of an individual, e.g., a non-cancerous cell suchas a non-cancerous APC, and cause the generation of an immune response,e.g., a humoral and/or cellular immune response, against another cell ofthe individual, e.g., a cancer cell.

[0126] Some individuals exposed to high levels of carcinogens, such assmokers, may express high levels of CYP1B1 and therefore displaytolerance to the protein. By generating an anti-CYP1B1 immune responseby methods described herein, this tolerance may be broken in such anindividual, thereby resulting in the generation of an immune responseagainst a CYP1B1-expressing cell, e.g., a cancer cell.

[0127] The nucleic acid constructs described herein can also be used inex vivo treatment. For example, cells such as dendritic cells,peripheral blood mononuclear cells, or bone marrow cells can be obtainedfrom an individual or an appropriate donor and activated ex vivo with anucleic acid composition or polypeptides encoded by the nucleic acidsdescribed herein, and then returned to the individual. In addition,cells such as myoblasts can be transfected or infected with a nucleicacid expression vector described herein, and then administered to anindividual. The CYP1B1 -expressing myoblasts can thus act as an in vivosource of CYP1B1 for generating an anti-CYP1B1 immune response.

[0128] The methods described herein for generating an anti-CYP1B1 immuneresponse can also include generating an immune response against one ormore additional cancer related antigens, such as telomerase,carcinoembryonic antigen (CEA), or p53, incident to the generation of ananti-CYP1B1 immune response. Such an immune response can be achieved bya variety of methods, including administration of a nucleic acidencoding two polypeptides, wherein the first polypeptide is a CYP1B1-containing polypeptide described herein and the second is a polypeptidecontaining all or a portion of a cancer related antigen.

[0129] In some embodiments, the polypeptides encoded by the constructscan be used to enhance the immune response or break self tolerance viaan indirect approach. Nucleic acids and polypeptides encoded by them cancontain an immunostimulatory agent which would result in stimulation oflocal or systemic inflammatory responses. An example of insertingpeptide epitopes into proteins to make the resulting protein immunogenicis found in WO 95/05849, herein incorporated by reference.

[0130] The effect of nucleic acids and the encoded polypeptides can beenhanced with a prime (e.g., a plasmid, viral vector, or bacterialvector or polypeptide) followed by a boost of the same material, to helpenhance the immune response (see, e.g., WO 98/56919).

[0131] Nucleic acids or polypeptides encoded by the nucleic acids of theinvention can be used to monitor tumor development in murine tumormodels (Table 2). Mice are immunized with 50-100 ug of a plasmidcontaining a nucleic acid of the invention one to three times at threeweek intervals. The plasmid can be delivered in a microparticle or otherdelivery vehicle, or may be delivered as naked DNA. Then the tumor cellsare implanted into mice. At a time post-immunization (varies forindividual tumors), tumor development is monitored by assessing tumorgrowth or activity relative to its initial growth or activity. Adetermination is made that the tumor has either been slowed or inhibitedin its growth or has decreased in activity. The experiment can also beperformed by administering the tumor prior to immunization with theCYP1B1-encoding nucleic acid to demonstrate the therapeutic effects ofthe nucleic acid formulation. TABLE 2 Tumor/Strain Combinations TumorType Host MHC P815 Mastocytoma DBA/2 H-2^(d) Clone M3 Melanoma DBA/2H-2^(d) (Cloudman S91) CT26.WT Colon carcinoma BALB/c H-2^(d) A20 B celllymphoma BALB/c H-2^(d) J558 Plasmacytoma BALB/c H-2^(d) EL4 ThymomaC57BL/6 H-2^(b) B16/F10 Melanoma C57BL/6 H-2^(b) 3LL Lung CarcinomaC57BL/6 H-2^(b) Sa1 Fibrosarcoma A/J H-2^(a(k/d))

[0132] The invention also includes methods of stimulating an immuneresponse in a mammal belonging to a first species by administering tothe mammal, e.g., a human or a mouse, a nucleic acid encoding apolypeptide containing a CYP1B1 polypeptide or portion thereof thatbinds to an MHC class I or class II molecule or immunoglobulin receptor.In these methods, the CYP1B1 polypeptide or MHC-binding portion orimmunoglobulin binding portion thereof is identical to a sequence of anaturally occurring CYP1B1 polypeptide of a second species, e.g., arodent such as a mouse or rat. The nucleic acid used in this method canoptionally be a nucleic acid of the invention, e.g., a nucleic acid thatlacks sequences found in the untranslated region (UTR) of naturallyoccurring forms of a CYP1B1 transcript, or a nucleic acid encoding aCYP1B1 with mutations, deletions, insertions, or rearrangements. Thenucleic acid can be administered to the mammal naked or via any of thedelivery vehicles described herein, e.g., in a microparticle or apolymeric hydrogel matrix. Delivery of a nucleic acid to a first speciesas described above may result in the development of a herteocliticimmune response in the first species, e.g., an immune response directedto CYP1B1 sequences endogenously produced by the first species. Thismethod could therefore be used to break T cell tolerance and induce aCYP1B1 T cell response in the first species. In these methods, themammal may have or be at risk of developing a cellular proliferativedisorder, e.g., cancer.

[0133] Delivery of Nucleic Acids

[0134] The compositions of the invention may be used to deliver, intoappropriate cells, nucleic acids that express peptides intended tostimulate an immune response against various cancers. An advantage ofgene delivery is that the antigenic peptide can be produced inside thetarget cell itself, where the interaction with a MHC molecules to whichthe immunogenic peptide binds is kinetically favored. This is incontrast to some vaccine protocols that do not specifically directantigenic peptides to MHC molecules. Alternatively, the polypeptide canbe secreted, resulting in the activation of immune cells such as Bcells.

[0135] The nucleic acids of the invention can be administered usingstandard methods, e.g., those described in Donnelly et al., J. Imm.Methods 176:145, 1994, and Vitiello et al., J. Clin. Invest. 95:341,1995. Nucleic acids of the invention can be injected into subjects inany manner known in the art, e.g., intramuscularly, intravenously,intraarterially, intradermally, intraperitoneally, intranasally,intravaginally, intrarectally or subcutaneously, or they can beintroduced into the gastrointestinal tract, the mucosa, or therespiratory tract, e.g., by inhalation of a solution or powdercontaining microparticles. Alternately, the compositions of theinvention may be applied to the skin or electroporated into cells ortissue, either in vitro or in vivo. Alternately, the compositions of theinvention may be treated with ultrasound to cause entry into the cellsor tissue. Additionally, the compositions of the invention may beadministered via a gene gun. Long lasting continuous release of thepolypeptides, analogs or nucleic acids of the invention can also beobtained, for example, through the use of osmotic pumps. Administrationcan be local (e.g., intramuscularly or at the tumor or other site ofinfection) or systemic.

[0136] The nucleic acids can be delivered in a pharmaceuticallyacceptable carrier such as saline, lipids, liposomes, microparticles, ornanospheres, as hydrogels, as colloidal suspensions, or as powders. Theycan be naked or associated or complexed with delivery vehicles and/ortransfection facilitating agents and delivered using delivery systemsknown in the art, such as lipids, liposomes, microspheres,microparticles, microcapsules, gold, nanoparticles, polymers, condensingagents, polysaccharides, polyamino acids, dendrimers, saponins,adsorption enhancing materials, membrane permeabilizing agents such asstreptolysin O, or fatty acids. Examples of hydrogel networks aredescribed in U.S. Ser. No. 60/270,256, filed Feb. 20, 2001.

[0137] The nucleic acids can include nuclear localization signals thatpromote the translocation of the nucleic acid to the nucleus. Forexample, a nucleic acid can include a sequence of nucleotides that isbound by a DNA binding protein, such as a transcription factor. Inanother example, a peptide based nuclear localization signal can beprovided with a nucleic acid of the invention, to thereby promote thetranslocation of the nucleic acid to the nucleus. Examples of usefulsignals include hnRNPA sequences and the SV40 nuclear localizationsignal. A nuclear localization peptide sequence can be, for example,mixed with a nucleic acid, conjugated to a nucleic acid, or incorporatedin a delivery vehicle such as a liposome or microparticle.

[0138] Other standard delivery methods, e.g., biolistic transfer, or exvivo treatment, can also be used. In ex vivo treatment, e.g., antigenpresenting cells (APCs), dendritic cells, peripheral blood mononuclearcells, or bone marrow cells can be obtained from a patient or anappropriate donor and activated ex vivo with the immunogeniccompositions, and then returned to the mammal.

[0139] Microparticles, including those described in U. S. Pat. No.5,783,567 and U.S. Ser. No. 60/208,830, filed Jun. 2, 2000, can be usedas vehicles for delivering macromolecules such as DNA, RNA, orpolypeptides into cells. They may therefore be useful for deliveringnucleic acids described herein, optionally with immunostimulatoryagents, to a cell of an individual. Microparticles containmacromolecules embedded in a polymeric matrix or enclosed in a shell ofpolymer. Microparticles act to maintain the integrity of themacromolecule, e.g., by maintaining the enclosed DNA in a nondegradedstate. Microparticles can also be used for pulsed delivery of themacromolecule, and for delivery at a specific site or to a specific cellor target cell population such as macrophages, monocytes, or dendriticcells. Microparticle formulations can also be used to activate relevantcell populations such as macrophages, monocytes or dendritic cells.

[0140] The polymeric matrix can be a biodegradable co-polymer such aspoly-lactic-co-glycolic acid, starch, gelatin, or chitin. Microparticlescan be used in particular to maximize delivery of DNA molecules into asubject's phagocytotic cells. Alternatively, the microparticles can beinjected or implanted in a tissue, where they form a deposit. As thedeposit breaks down, the nucleic acid is released gradually over timeand taken up by neighboring cells (including APCS) as free DNA.

[0141] Microparticles may also be formulated as described by Mathiowitzet al. (WO 95/24929) and U.S. Pat. Nos. 5,817,343 and 5,922,253, hereinincorporated by reference.

[0142] The nucleic acids of the invention can be administered intosubjects via lipids, dendrimers, or liposomes using techniques that arewell known in the art. For example, liposomes carrying immunogenicpolypeptides or nucleic acids encoding immunogenic peptides are known toelicit CTL responses in vivo (Reddy et al., J. Immunol. 148:1585, 1992;Collins et al., J. Immunol. 148:3336-3341, 1992; Fries et al., Proc.Natl. Acad. Sci. USA 89:358, 1992; Nabel et al., Proc. Nat. Acad. Sci.(USA) 89:5157, 1992).

[0143] The nucleic acids of the invention can be administered by usingImmune Stimulating Complexes (ISCOMS), which are negatively chargedcage-like structures 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A (saponin), or saponin alone. The peptides andnucleic acid of the invention can be co-administered with the ISCOMS, orcan be administered separately.

[0144] Protective immunity has been generated in a variety ofexperimental models of infection, including toxoplasmosis andEpstein-Barr virus-induced tumors, using ISCOMS as the delivery vehiclefor antigens (Mowat et al., Immunology Today 12:383-385, 1991). Doses ofantigen as low as 1 Fg encapsulated in ISCOMS have been found to produceclass I-mediated CTL responses, where either purified intact HIV-1 -IIIBgp 160 envelope glycoprotein or influenza hemagglutinin is the antigen(Takahashi et al. (1990) Nature 344:873).

[0145] It is expected that a dosage of approximately 1 to 200 μg of DNAwould be administered per kg of body weight per dose. Where the patientis an adult human, vaccination regimens can include, e.g.,intramuscular, intravenous, oral, intranasal, intrarectal, orsubcutaneous administrations of 10-1000 μg of DNA when delivered in amicroparticle or other delivery vehicle, or of about 1-18 mg of nakedDNA delivered intramuscularly or intradermally, repeated 1 -12 times. Ofcourse, as is well known in the medical arts, dosage for any givenpatient depends upon many factors, including the patient's size, bodysurface area, age, sex, and general health; the time and route ofadministration; the particular compound to be administered; and otherdrugs being administered concurrently. Determination of optimal dosageis well within the abilities of a pharmacologist of ordinary skill.

[0146] Measuring Responses of the Immune System to the Nucleic Acids

[0147] The ability of nucleic acids described herein to elicit an immuneresponse can be assayed by using methods for measuring immune responsesthat are well known in the art. For example, the generation of cytotoxicT cells can be demonstrated in a standard ⁵Cr release assay, bymeasuring intracellular cytokine expression, or by using MHC tetramers.Standard assays, such as ELISA or ELISPOT, can also be used to measurecytokine profiles attributable to T cell activation. T cellproliferation can also be measured using assays such as ³H-thymidineuptake and other assays known in the art. DTH responses can be measuredto assess T cell reactivity. B cell responses can be measured using artrecognized assays such as ELISA.

[0148] Other methodologies, e.g., digital imaging and cytologic,colposcopic and histological evaluations, can also be used to evaluatethe effects of immunogenic peptides, and of nucleic acid encoding theimmunogenic peptides, on various types of proliferative disease orcancer.

[0149] The following are examples of the practice of the invention. Theyare not to be construed as limiting the scope of the invention in anyway.

EXAMPLES EXAMPLE 1: Generation of human CYP1B1 cDNA constructs

[0150] cDNAs encoding human CYP1B1 (SEQ ID NO:2) and CYP1B1 -delta3 (SEQID NO:38) were each cloned into two different plasmid expressionvectors, pCDNA-3 and p3K. The CYP1B1 nucleic acid constructs contained acDNA coding for a 543 amino acid protein, but lacking all untranslatedregions of CYP1B1. The CYP1B1 -delta3 construct contained threesubstitutions, relative to the wild type CYP1B1 of SEQ ID NO:2, at aminoacid positions 57, 61 and 365 (amino acids 57: Trp changed to a Cys;amino acid 61: Gly changed to a Glu; amino acid 365: Gly changed to aTrp; see SEQ ID NO:38). The expression vectors pcDNA3-CYPHu1B1,p3k-CYPHu1B1, pcDNA3-CYPHu1B1-delta 3 (pcDNAhu1B1d3), p3K-CYPHu1B1-delta3 (p3khu1B1d3), and control vectors pcDNA3 and p3K were purified fromtransformed Esherichia coli using Qiagen columns according to themanufacturers instructions (Qiagen, Chatsworth, Calif.). Each constructwas sequenced to confirm the introduction of the desired changes.

[0151] Additional CYP1B1 constructs were made as follows. Deletions wereintroduced using PCR, in the background of pcDNA3hu1B1d5. pcDNA3hu1B1d5encodes a human CYP1B1 protein in which five amino acids aresubstituted: W57C, G61E, G365W, P379L, and E387K. Upstream primerscontained a restriction site and an ATG codon in frame with thesubsequent coding sequences. Downstream primers contained theappropriate CYP1B1 coding sequences, followed by the stop codon, and arestriction site for cloning purposes. pcDNA3hu1B1-deltaPPGP encodes thewhole CYP1B1 protein, with the exception of amino acids 51 to 54 (PPGP),which were deleted. The pcDNA3hu1B1-F1R1-encoded protein contains adeletion of the first 60 amino acids of CYP1B1, and the pcDNA3hu1B1-F1R2protein contains the same N-terminal deletion, in addition to the last82 amino acids of the CYP1B1 protein. The pcDNA3hu1B1-F2R1 proteinencompasses a deletion of the first 171 amino acids of CYP1B1, and thepcDNA3hu1B1-F2R2-encoded protein contains the same N-terminal deletion,in addition to the last 82 amino acids of CYP1B1 . The pcDNA3hu1B1-F3R1protein contains a deletion of the first 292 amino acids of CYP1B1, andpcDNA3hu1B1-F3R2 contains the same N-terminal deletion, in addition tothe last 82 amino acids of the CYP1B1 protein (FIG. 3). The DoublePEP-Padre protein is depicted as SEQ ID NO:41 in FIG. 3.

[0152] The HLA-A2K^(b) transgenic C57B1/6 mouse line produces a hybridMHC class I molecule. In this hybrid molecule, the peptide bindingdomains (alphal and alpha2) are derived from the human class I moleculeHLA-A*0201, whereas the domain (alpha3) which interacts with the CD8co-receptor on T cells is derived from the murine class I moleculeK^(b). The resulting animal is capable of responding to immunogens whichcontain HLA-A2 restricted epitopes and of generating murine cytotoxic Tcells (CTLs) that recognize human target cells expressing HLA-A2(Vitiello et al., J. Exp. Med. 173:1007, 1991).

[0153] The experiments described in the following examples demonstratedthat: 1) plasmids encoding CYP1B1 were expressed; 2) the CYP1B1 proteinwas translated; 3) the encoded proteins were processed and the peptidespresented on class I receptors in vivo; 4) and the T cell repertoirecontained cells capable of recognizing the class I/peptide complex.

EXAMPLE 2: Immunization of mice with DNA expression vector encoding wildtype CYP1B1 elicits T cell immunitv in HLA-A2 transgenic mice in vivo

[0154] Groups of at least three 6-8 week old female transgenic mice,expressing the human Class I molecule HLA-A2.1 were immunized withpcDNA3-CYPHulB1 or pcDNA3 plasmid vectors. 100 micrograms of plasmid DNAwas injected into each anterior tibialis muscle. A booster immunizationwas performed 14 days after the first immunization. Twelve daysfollowing the second immunization, splenocytes were harvested.

[0155] Single cell suspension of spleens from two to three mice wereprepared in RPMI-1640 medium with 10% fetal calf serum and antibiotics.Red blood cells were lysed by incubation of the cells in 0.83% NH4Cl at4° C for 10 minutes. After washing, the cells were separated on a CD8 Tcell enrichment column according to the manufacturer's protocol (MurineT cell CD8 Subset column kit, R&D System, Minneapolis, Minn.). A mouseinterferon-gamma (IFN-g) enzyme-linked immunospot (ELISpot) assay, wasused for the detection of individual human CYP1B1 epitope specific CD8+T cells (Mouse IFN-g ELISpot, R&D Systems). Briefly 1×10⁵ purifiedspleen cells were incubated in vitro with equal number of syngeneicEL-4-A2/Kb target cells, which had been pre-pulsed with 10 micromolesynthetic HLA-A2.1 binding peptide, FLDPRPLTV (SEQ ID NO:22) or infectedwith a human CYP1B1 -expressing vaccinia vector for 14 hours. After 24hours of co-culture, T cell activity was determined by performing theELIspot assay according to the manufacturer's instructions (Mouse IFN-gELISpot, R&D Systems). The spots, representing the frequency of CYP1B1reactive T cells, were counted with an automated ELISpot reader system(FIG. 4).

EXAMPLE 3: A CYP1B1 variant cDNA construct elicits T cell immunity inHLA-A2 transgenic mice in vivo

[0156] Groups of at least three 6-8 week old female transgenic mice,expressing the human Class I molecule HLA-A2.1 were immunized withp3K-CYPHu1B1-delta 3 or p3K plasmid vectors. 100 micrograms of plasmidDNA was injected into each anterior tibialis muscle. A boosterimmunization was performed 14 days after the first immunization. Twelvedays following the second immunization, splenocytes were harvested.

[0157] Single cell suspension of spleens from two to three mice wereprepared in RPMI-1640 medium with 10% fetal calf serum and antibiotics.Red blood cells were lysed by incubation of the cells in 0.83% NH4Cl at4° C. for 10 minutes. After washing the cells were separated on a CD8 Tcell enrichment column according to the manufacturer's protocol (MurineT cell CD8 Subset column kit, R&D System, Minneapolis, Minn.). A mouseinterferon-gamma (IFN-g) enzyme-linked immunospot (ELISpot) assay, wasused for the detection of individual human CYP1B1 epitope specific CD8+T cells (Mouse IFN-g ELISpot, R&D Systems). Briefly 1×10⁵ purifiedspleen cells were incubated in vitro with equal number of syngeneicEL-4-A2/Kb target cells, which had been pre-pulsed with 10 micromolesynthetic HLA-A2.1 biding peptide, FLDPRPLTV (SEQ ID NO:22) or infectedwith a human 1B1 expressing vaccinia vector for 14 hours. After 24 hoursof co-culture, T cell activity was determined by performing the ELIspotassay according to the manufacturer's instructions (Mouse IFN-g ELISpot,R&D Systems). The spots, representing the frequency of CYP1B1 reactive Tcells, were counted with an automated ELISpot reader system (FIG. 5).

EXAMPLE 4: Detection of Anti-CYP1B1 Antibodies by Western blot analysis

[0158] Serum of an animal immunized with a CYP1B1 encoding nucleic acidcan be tested for the presence of anti-CYP1B1 antibodies, as follows.Human CYP1B1 microsomes (Gentest, Woburn Mass.) containing 30 ug ofmicrosomal protein are boiled in SDS sample buffer (Boston Bioproducts,Ashland, Mass.) and electrophoretically separated on 10% Tris-HClacrylamide gels (Bio-Rad, Chicago, Ill.). The gel is electroblotted ontonitrocellulose (Bio-Rad). Non-specific protein binding sites are blockedby incubation of nitrocellulose membranes for 60 minutes at roomtemperature with 5% non-fat milk in TBST buffer (50 mM Tris [pH 8], 150mM NaCl, and 0.05% Tween-20). Nitrocellulose filters are incubated witheither variable dilutions of immune mouse test serum (e.g., from miceimmunized with CYP1B1 peptides or nucleic acid) or a 1:40 to 1:3000dilution of mouse anti human CYP1B1 443-457 peptide 5D3 mAb in hybridomaculture supernatant diluted in TBST-5% non-fat milk. The membrane isthen incubated with a 1:2000 dilution of goat anti-mouse-horseradishperoxidase antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)diluted in TBST 5% non-fat skim milk. After incubation with eachantibody the membrane is washed for five 10 minute periods in TBST. Themembrane is developed with ECL reagent (Amersham Pharmacia Biotech,Uppsala, Sweden) to demonstrate the presence of specific protein bands.

EXAMPLE 5: Electroporation of CYP1B1 nucleic acid constructs

[0159] The effect of electroporation on the T cell response induced in aA2/K^(b) transgenic mouse following a single immunization withp3khu1B1d3 DNA was investigated. Mice were injected with p3khu1B1d3, p3Kcontrol vector, or were untreated. Treatment groups were subsequentlydivided and treated by electroporation or subjected to no furthertreatment. Electroporation significantly increased the frequency of Tcells reactive against EL4-A2/K^(b) cells either pulsed with CYP 190peptide or infected with recombinant vaccinia virus expressing thenative CYP1B1 protein.

[0160]FIG. 6 shows that T cell reactivity in HLA-A2/K^(b) transgenicmice is enhanced by electroporation. Paired groups of mice wereimmunized once with p3khu1B1d3 DNA, p3K control DNA, and one group pertreatment was subjected to electroporation (++) or left untreated. CD8+T-enriched spleen cells were tested in a direct IFN-γ ELISpot assayagainst EL4-A2/K^(b) cells either pulsed with human peptide CYP190(CYP190) or left untreated (untreated). Antigen-specific T-cellfrequencies are reported as spot forming cells/10e6 CD8+ T cells.

[0161] HLA-A2/K^(b) transgenic female mice 6-8 weeks of age were used.Plasmid DNA for injection was made with endotoxin-free plasmidpurification kits according to the manufacturer's instructions (QIAGENInc., Chatsworth, Calif.). A 25 μl volume was injected into the tibialisanterior muscle of each leg for a total dose of 100 μg of DNA.Electroporation was performed on anesthetized mice immediatelythereafter by intramuscular insertion of a BTX needle array (Model 532)across the DNA injection site and delivery of pulses (100V, 20 msecpulse length×8) by an ElectoSquarePorator Model T820 (Genetronics).Animals were immunized once (two mice per group) and assayed 12 dayslater. Murine CD8+ T-cell responses to CYP1B1 were analyzed by IFN-γELISPOT using a commercial IFN-γ ELISPOT assay kit according to themanufacturer's recommendations (R&D Systems, Minneapolis, Minn.). Aseffector cells, pooled spleen cells were enriched for CD8⁺ T-cells(Murine T Cell CD8 Subset column Kit; R&D Systems) and plated induplicate at 1×10⁵ cells/well. T-cells were stimulated with 1×10⁵EL4-A2/K^(b) cells/well pulsed with 10 ug/ml peptide or infected withrecombinant vaccinia virus or wt vaccinia for 16-18 hours prior toplating (MOI, 10). Plates were incubated for 24 hours, developed, andanalyzed by automated image analysis (Zellnet Consulting, Inc., NewYork, N.Y.). Antigen-specific T-cell frequencies are reported as spotforming cells (SFC)/1×10⁶ CD8⁺ T-cells. The murine thymoma cell line EL4was obtained from ATCC (Manassas, Va.) and was transfected with theHLA-A2/K^(b) cDNA inserted into the pSV2neo vector. The human CYP1B1peptide CYP190 (FLDPRPLTV; SEQ ID NO:22) was purchased from HarvardMedical School Biopolymers Laboratory (Boston, Mass.).

EXAMPLE 6. Hydrogels and microparticles containing CYP1B1 nucleic acidconstructs

[0162] Delivery of CYP1B1 d3 DNA by different delivery systems canelicit specific immune responses in HLA-A2/K^(b) transgenic mice.HLA-A2/K^(b) transgenic mice were used to evaluate if DNA delivered in ahydrogel could generate an immune response in vivo. Administration of 3doses of 2% polymeric network, hydrogel formulation or microparticlescontaining a DNA construct encoding a mutated CYP1B1 cDNA(pcDNA3hu1B1d3) elicited a high frequency of splenic CD8+ T cells withspecific reactivity against EL4-A2/K^(b) cells either pulsed with theHLA-A2 restricted CYP190 peptide or infected with a recombinant vacciniavirus encoding the full length, native huCYP1B1 CDNA. Significantreactivity was not observed against either EL4-A2/K^(b) cells leftuntreated or infected with wild type vaccinia virus. Animals immunizedwith formulated vector (pcDNA 3) DNA did not elicit a response.

[0163]FIG. 7 shows the induction of a CYP1B1 -specific T cell responsein A2/K^(b) transgenic mice by immunization with a CY1B1 DNA/hydrogelformulation. HLA-A2 transgenic mice were immunized 3 times with pcDNA3DNA or pcDNA3hu1B1d3 delivered in either PLG microparticles or 2%polymeric network hydrogels. CD8+ T-enriched spleen cells were tested ina direct IFN-γ ELISpot assay against EL4-A2/K^(b) cells either pulsedwith human peptide CYP 190 (CYP 190) or infected with vaccinia virusencoding CYP1B1 (vacHu1B1). Untreated (untreated) as well as, vacciniawild type-infected EL4-A2/Kb cells (Vac wt) were included as controls.Antigen-specific T-cell frequencies are reported as spot forming cells(SFC)/1×10⁶ CD8⁺ T-cells.

[0164] Female mice 6-8 weeks of age were used in all experiments.Plasmid DNA for injection was made with endotoxin-free plasmidpurification kits according to the manufacturer's instructions (QIAGENInc., Chatsworth, Calif.). A 25 μl volume was injected into the tibialisanterior muscle of each leg for a total dose of 100 μg of DNA. Theplasmid DNA was encapsulated into microparticles or formulated intopolymeric network hydrogels as described herein and used forimmunization. Animals were immunized three times at biweekly intervals(two mice per group) and assayed 12 days after last immunization.Microparticles were administered in 3 muscles/6 sites per animal:tibialis, calf (soleus muscle),and thigh. Polymeric network hydrogelswere injected as 100 ug DNA/100 ul volume; 50 ul per tibialis. MurineCD8+ T-cell responses to CYP1B1 were analyzed by IFN-γ ELISPOT using acommercial IFN-γ ELISPOT assay kit according to the manufacturer'srecommendations (R&D Systems, Minneapolis, Minn.). As effector cells,pooled spleen cells were enriched for CD8⁺ T-cells (Murine T Cell CD8Subset column Kit; R&D Systems) and plated in duplicate at 1×10⁵cells/well. T-cells were stimulated with 1×10⁵ EL4-A2/K^(b) cells/wellpulsed with 10 ug/ml peptide or infected with recombinant vaccinia virusor wt vaccinia for 16-18 hours prior to plating (MOI, 10). Plates wereincubated for 24 hours, developed, and analyzed by automated imageanalysis (Zellnet Consulting, Inc., New York, N.Y.). Antigen-specificT-cell frequencies are reported as spot forming cells (SFC)/1×10⁶ CD8⁺T-cells.

EXAMPLE 7: Microparticles containing CYP1B1 nucleic acid constructs

[0165] HLA-A2/K^(b) transgenic mice were used to evaluate whether DNAencapsulated in PLG microparticles could generate an immune response invivo. Administration of two doses of microparticles containing a DNAconstruct encoding a mutated CYP1B1 cDNA (p3khu1B1d3; ZYC300) elicited Tcells with specific reactivity against EL4-A2/K^(b) cells infected witha recombinant vaccinia virus encoding the full length, native huCYP1B1cDNA. Significant reactivity was not observed against EL4-A2/K^(b) cellsinfected with wild-type vaccinia virus. Neither non-immunized animalsnor animals immunized with a vector control showed an IFN-γ response toeither stimulator.

[0166] Induction of effective anti-tumor immunity involves expansion ofan effector T-cell repertoire against self antigens. Data suggesting theeffectiveness of CYP1B1 as a therapeutic tumor antigen in thistransgenic mouse model would demonstrate that a T-cell response to mouseCYP1B1 self determinants in the context of the endogenous murine class IMHC are elicited following immunization. Mouse and human CYP1B1 sharesignificant sequence homology at the amino acid level. Using thepreviously described peptide-binding algorithms, a predicted H-K^(b)binding CYP1B1 peptide was identified as a reagent to test for inductionof mouse-specific self responses. This CYP1B1 residue 77-84 (LARRYGDV;SEQ ID NO:42) is shared between human and mouse orthologs. The peptidewas included together with the human CYP190 HLA-A2 epitope to test forits ability to detect responses in mice immunized with microparticlescontaining human CYP1B1 DNA. As shown, a clear response could bedetected against this peptide.

[0167]FIG. 8 shows that immunization of mice with encapsulated DNAencoding CYP1B1 elicits CYP1B1 -specific immune responses in transgenicmice. HLA-A2/K^(b) transgenic mice were not immunized (left bars), orwere immunized twice with microparticles containing p3khu1B1d3 (rightbars) or p3k control (center bars). CD8⁺ enriched spleen cells weretested for immune response against EL4-A2K^(b) tumor cells in a directELISpot assay. Target cells were pulsed with HLA-A2 peptides CYP190,peptide CYP77, or were infected with either CYP1B1 -vaccinia (VacHu1B1)or vaccinia wild-type control (Vac Wt).

[0168] HLA-A2/K^(b) transgenic mice were immunized as described abovewith endotoxin-free plasmid encapsulated in PLG microparticles into thetibialis anterior muscle of each leg for a total dose of 100 μg of DNA.Animals were immunized at two-week intervals and assayed 12 days afterthe last immunization. Murine CD8⁺ T cell responses to CYP1B1 wereanalyzed by IFN-γ ELISpot assay. Effector spleen cells, were enrichedfor CD8⁺ T-cells and plated in duplicate at 1×10⁵ cells/well. T cellswere stimulated with 1×10⁵ EL4-A2K^(b) cells/well pulsed with 10 μg/mlpeptide or infected with recombinant vaccinia virus or wt vaccinia for16-18 hours prior to plating (MOI, 10). Antigen-specific T-cellfrequencies are reported as spot forming cells/1×10⁶ CD8⁺ T-cells.Peptide CYP77 (LARRYGDV; SEQ ID NO:42) is shared between human and mouseCYP1B1 DNA sequence and was purchased from Multiple Peptide Systems (SanDiego, Calif.).

EXAMPLE 8: MHC class II responses in mice injected with CYP1B1 nucleicacid constructs

[0169] Experiments were performed to evaluate whether pcDNA3-hu1B1encodes a protein that is processed, presented, and can stimulate MHCclass II CD4+ T cell responses in multiple strains of inbred mice. ClassII CD4+ T cell responses were detected using an ex-vivo IFN-g Elispotassay with synthetic peptides derived from the CYP1B1 protein.

[0170] Three strains of mice (C3H, C57/B16 and Balb/c) were injected(intramuscularly) with 100 μg of pcDNA3-hu1B1. Mice were boosted(intramuscularly) on day 14 with the same dose ofpcDNA3-hu1B1. Spleenswere harvested on day 27 and IFN-g ELISPOT assays were performed usingCD4+ T cell enriched splenocytes tested against syngeneic APC pulsedwith peptide. In addition, CD4+ T cells isolated from naive mice werescreened to serve as a negative control. All CD4+ T cells were screenedagainst a panel of synthetic CYP1B1 30 mer peptides (see Table 3 forsequences), PHA (positive assay control), and HBV-2 (negative assaycontrol). TABLE 3 Synthetic CYP1B1 Peptides 1B1 SEQ ID Peptide #Sequence NO 2 RQRRRQLRSAPPGPFAWPLIGNAAAVGQAA 43 3HLSFARLARRYGDVFQIRLGSCPIVVLNGE 44 4 RAIHQALVQQGSAFADRPAFASFRVVSGGR 45 5SMAFGHYSEHWKVQRRAAHSMMRNFFTRQP 46 6 RSRQVLEGHVLSEARELVALLVRGSADGAF 47 8GCRYSHDDPEFRELLSHNEEFGRTVGAGSL 48 9 FGRTVGAGSLVDVMPWLQYFPNPVRTVFRE 49 10FEQLNRNFSNFILDKFLRHCESLRPGAAPR 50 12 WLLLLFTRYPDVQTRVQAELDQVVGRDRLP 5113 CMGDQPNLPYVLAFLYEAMRFSSFVPVTIP 52 14 HATTANTSVLGYHIPKDTVVFVNQWSVNHD53 16 IGEELSKMQLFLFISILAHQCDFRANPNEP 54 17KSFKVNVTLRESMELLDSAVQNLQAKETCQ 55

[0171] Table 4 depicts the results of the above assay for those CYP1B1peptides that stimulated a response in each of the three mouse strainstested. All reported values IFN-g Spot Forming Cells (SFC) 1,000,000CD4+ T cells. TABLE 4 MHC class II response in C3H, C57/B16, and Balb/cMice C3H mice receiving Media HBV-2 PHA Peptide #9 Peptide #10pcDNA3-hu1B1 4 0 78 22 48 Naïve C3H mice 0 0 148 6 6 C57/B16 micereceiving Media HBV-2 PHA Peptide #2 Peptide #13 Peptide #17pcDNA3-hu1B1 0 4 28 44 196 176 Naïve C57/B16 mice 0 4 24 8 2 0 Balb/cmice receiving Media HBV-2 PHA Peptide #6 Peptide #9 Peptide #12pcDNA3-hu1B1 8 2 158 204 20 28 Naïve Balb/c mice 0 2 110 2 4 0

EXAMPLE 9: Immunization with deletion constructs of CYP1B

[0172] To examine if CYP1B1 can be altered to yield an effectiveimmunogen, a series of CYP1B1 cDNAs were engineered in which progressiveportions of the N- and C-termini were deleted. The constructs werecloned into the p3khu1B1d5 background, and hence contained the 5 pointmutations of this construct. To verify expression, the constructs weretransfected into 293 T cells (ATCC) and two days later lysates weregenerated from the transfected cells. The lysates were analyzed bySDS-PAGE and a Western analysis was performed on the transferred gel.The blot was probed with a monoclonal antibody specific for CYP1B1 anddetection was via enhanced chemiluminescence (ECL kit, Amersham). Thedata from this experiment demonstrated that the variant CYP1B1 proteinswere expressed from the deletion constructs.

[0173] To determine if the constructs induced CYP1B1-specific T cells,HLA-A2/K^(b) transgenic mice were immunized with DNA constructs encodingthe truncated CYP1B1 cDNAs. FIG. 9 shows that CYP1B1-specific T-cellswere induced by immunization with truncated CYP1B1 DNA constructs.HLA-A2 transgenic mice were untreated or were immunized with cDNAconstructs encoding mutated and/or truncated forms of the CYP1B1protein. CD8⁺ T-enriched spleen cells from immunized mice were tested ina direct IFN-g ELISpot assay against EL4-A2/K^(b) cells either pulsedwith human peptide CYP190 (CYP 190), mouse peptide CYP373 (mCYP373), orinfected with wild type vaccinia (Vac wt) or vaccinia virus encodingCYP1B1 (VacHu1B1). Untreated as well as vaccinia wild type-infectedEL4-A2/K^(b) cells were included as controls. Antigen-specific T-cellfrequencies are reported as spot forming cells (SFC)/1×10⁶ CD8⁺ T-cells.Mice were immunized with DNA encoding the indicated construct.

[0174] Female mice 6-8 weeks of age were used in all experiments.Plasmid DNA for injection was made with endotoxin-free plasmidpurification kits according to the manufacturer's instructions (QIAGENInc., Chatsworth, Calif.). A 25 μl volume was injected into the tibialisanterior muscle of each leg for a total dose of 100 μg of DNA. Animalswere immunized at days 0 and 14 (two mice per group) and assayed 12 daysafter last immunization. Murine CD8⁺ T-cell responses to CYP1B1 wereanalyzed by IFN-g ELISPOT using an assay kit according to themanufacturer's recommendations (R&D Systems, Minneapolis, Minn.). Aseffector cells, pooled spleen cells were enriched for CD8⁺ T-cells(Murine T Cell CD8 Subset column Kit; R&D Systems) and plated induplicate at 1×10⁵ cells/well. T-cells were stimulated with 1×10⁵EL4-A2/K^(b) cells/well pulsed with 10 ug/ml peptide or infected withrecombinant vaccinia virus or wt vaccinia for 16-18 hours prior toplating (MOI, 10). Plates were incubated for 24 hours, developed, andanalyzed by automated image analysis (Zellnet Consulting, Inc., NewYork, N.Y.). Antigen-specific T-cell frequencies are reported as spotforming cells (SFC)/1×10⁶ CD8⁺ T-cells. Murine peptide CYP373(SDQQQPNLPYV; SEQ ID NO:56), was purchased from Multiple Peptide Systems(San Diego, Calif).

[0175] Other Embodiments

[0176] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not limit the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A nucleic acid comprising a transcriptional unitcomprising a coding sequence encoding a polypeptide comprising CYP1B1 ora portion thereof that comprises a peptide that binds to an MHC class Ior class II molecule, wherein the transcriptional unit does not containa translational repressor element operably linked to the codingsequence.
 2. The nucleic acid of claim 1, wherein the polypeptidecomprises a segment of CYP1B1 that is eight amino acids in length. 3.The nucleic acid of claim 2, wherein the polypeptide comprises SEQ IDNO:22:
 4. The nucleic acid of claim 3, wherein the polypeptide is lessthan 100 amino acids in length.
 5. The nucleic acid of claim 1, whereinthe transcriptional unit comprises an RNA stabilization sequence.
 6. Thenucleic acid of claim 1, wherein the nucleic acid comprises an induciblepromoter sequence operably linked to the transcriptional unit.
 7. Thenucleic acid of claim 1, wherein the polypeptide comprises a targetingsignal.
 8. A nucleic acid comprising a transcriptional unit comprising acoding sequence encoding a polypeptide comprising CYP1B1 or a portionthereof that comprises a peptide that binds to an MHC class I or classII molecule, wherein the transcriptional unit does not contain 150consecutive nucleotides of SEQ ID NO:18 or SEQ ID NO:19.
 9. The nucleicacid of claim 8, wherein the transcriptional unit does not contain atleast one of SEQ ID NOs:3-9 or 15-17.
 10. The nucleic acid of claim 9,wherein the transcriptional unit does not contain any of SEQ ID NOs:3-9or 15-17.
 11. The nucleic acid of claim 8, wherein the transcriptionalunit does not contain 50 consecutive nucleotides of SEQ ID NO:18 or SEQID NO:19.
 12. The nucleic acid of claim 8, wherein the transcriptionalunit does not contain 25 consecutive nucleotides of SEQ ID NO:18 or SEQID NO:19.
 13. The nucleic acid of claim 8, wherein the transcriptionalunit does not contain 10 consecutive nucleotides of SEQ ID NO:18 or SEQID NO:19.
 14. The nucleic acid of claim 8, wherein the transcriptionalunit comprises an RNA stabilization sequence.
 15. The nucleic acid ofclaim 8, wherein the nucleic acid comprises an inducible promotersequence.
 16. The nucleic acid of claim 8, wherein the transcriptionalunit comprises a translational regulatory sequence operably linked tothe coding sequence.
 17. The nucleic acid of claim 16, wherein thetranslational regulatory sequence is an iron responsive sequence. 18.The nucleic acid of claim 8, wherein the polypeptide comprises a segmentof CYP1B1 that is eight amino acids in length.
 19. The nucleic acid ofclaim 18, wherein the polypeptide comprises SEQ ID NO:22.
 20. Thenucleic acid of claim 19, wherein the polypeptide is less than 100 aminoacids in length.
 21. The nucleic acid of claim 8, wherein thepolypeptide comprises a targeting signal.
 22. A nucleic acid comprisinga transcriptional unit, wherein the transcriptional unit encodes ahybrid polypeptide comprising a first and a second segment of CYPB1,wherein the first and second segments are either contiguous or separatedby a spacer amino acid or spacer peptide, wherein the first and secondsegments are each at least eight amino acids in length, and wherein thefirst and second segments are non-contiguous portions of CYP1B1.
 23. Thenucleic acid of claim 22, wherein the hybrid polypeptide furthercomprises a third segment of CYP1B1 , wherein the third segment is atleast eight amino acids in length, and wherein the first and third andsecond and third segments are non-contiguous portions of CYP1B1 . 24.The nucleic acid of claim 22, wherein the first segment comprises thesequence of SEQ ID NO:22.
 25. The nucleic acid of claim 22, wherein thepolypeptide comprises a targeting signal.
 26. A composition comprisingthe nucleic acid of claim 1 and an immunostimulatory agent.
 27. Thecomposition of claim 26, wherein the immunostimulatory agent is a CpGcontaining oligonucleotide of 18-30 nucleotides in length.
 28. Thecomposition of claim 26, wherein the immunostimulatory agent is IL-12 orIFN-gamma.
 29. The composition of claim 26, wherein theimmunostimulatory agent is a lipid, nucleic acid, carbohydrate, orbacterial polypeptide.
 30. A composition comprising the nucleic acid ofclaim 1 and a nucleic acid encoding an immunostimulatory agent.
 31. Thecomposition of claim 30, wherein the immunostimulatory agent is IL-12,IFN-gamma, or a bacterial polypeptide.
 32. A therapeutic compositioncomprising the nucleic acid of claim 1 and a pharmaceutically acceptablecarrier.
 33. A microparticle comprising a polymeric matrix or shell andthe nucleic acid of claim
 1. 34. A microparticle comprising a polymericmatrix or shell and the nucleic acid of claim
 8. 35. A microparticlecomprising a polymeric matrix or shell and the nucleic acid of claim 22.36. A method of inducing an immune response in a mammal, comprisingadministering the nucleic acid of claim 1 to the mammal.
 37. The methodof claim 36, wherein the mammal suffers from or is at risk for cancer.38. The method of claim 36, wherein the nucleic acid is administeredsubcutaneously or intramuscularly.
 39. The method of claim 36, whereinthe immune response is directed to CYP1B1.
 40. The method of claim 39,wherein the immune response is a T cell response.
 41. The method ofclaim 39, wherein the immune response is a B cell response.
 42. A methodof inducing an immune response in a mammal, comprising administering themicroparticle of claim 33 to the mammal.
 43. A method of generating animmune response, the method comprising: detecting expression of CYP1B1in a tumor of a mammal; and administering the nucleic acid of claim 1 tothe mammal, wherein the administration results in the generation of ananti-CYP1B1 immune response in the mammal.
 44. A method of reducingtumor growth or tumor activity in a mammal, comprising: identifying amammal having a tumor; administering the nucleic acid of claim 1 to themammal; and detecting a reduction in the size or activity of the tumorfollowing the administration of the nucleic acid.
 45. The method ofclaim 44, further comprising detecting CYP1B1 expression in the tumorbefore administering the nucleic acid.
 46. A method of inducing animmune response in a mammal, the method comprising administering to amammal a nucleic acid encoding a polypeptide comprising CYP1B1 orportion thereof that binds to an MHC class I or class II molecule,wherein the mammal belongs to a first species, and wherein the CYP1B1 orportion thereof is identical to a sequence of a naturally occurringCYP1B1 polypeptide of a second species.
 47. The method of claim 46,further comprising identifying the mammal as having a tumor prior toadministering the nucleic acid to the mammal.
 48. The method of claim46, wherein the nucleic acid is the nucleic acid of claim
 1. 49. Themethod of claim 46, wherein the nucleic acid is the nucleic acid ofclaim
 22. 50. The method of claim 46, wherein the first species is ahuman.
 51. The method of claim 50, wherein the second species is arodent.
 52. The method of claim 51, wherein the rodent is a rat or amouse.